3′-OH unblocked, nucleotides and nucleosides, base modified with photocleavable, terminating groups and methods for their use in DNA sequencing

ABSTRACT

Provided are novel nucleotides, nucleoside, and their derivatives described herein, that can be used in DNA sequencing technology and other types of DNA analysis. In one embodiment, the nucleotide or nucleoside with an unprotected 3′-OH group is derivatized at the nucleobase to include a fluorescent dye attached via a linker to a photocleavable terminating group. The photocleavable-fluorescent group is designed to terminate DNA synthesis as well as be cleaved so that DNA oligomers can be sequenced efficiently in a parallel format. The design of such rapidly cleavable fluorescent groups on nucleotides and nucleosides can enhance the speed and accuracy of sequencing of large oligomers of DNA in parallel, to allow rapid whole genome sequencing, and the identification of polymorphisms and other valuable genetic information, as well as allowing further manipulation and analysis of nucleic acid molecules in their native state following cleavage of the fluorescent group.

This invention was made with government support under grant number R01HG003573-01 awarded by the NHGRI (National Human Genome ResearchInstitute), which is one of the institutes of the NIH. The governmenthas certain rights in this invention.

FIELD OF INVENTION

The present invention relates generally to compounds and methods for DNAsequencing and other types of DNA analysis. More particularly, theinvention relates to nucleotides and nucleosides labeled withphotocleavable groups and methods for their use in DNA sequencing andanalysis.

BACKGROUND

Methods for rapidly sequencing DNA have become needed for analyzingdiseases and mutations in the population and developing therapies. Themost commonly observed form of human sequence variation is singlenucleotide polymorphisms (SNPs), which occur in approximately 1-in-300to 1-in-1000 base pairs of genomic sequence. Building upon the completesequence of the human genome, efforts are underway to identify theunderlying genetic link to common diseases by SNP mapping or directassociation. Technology developments focused on rapid, high-throughput,and low cost DNA sequencing would facilitate the understanding and useof genetic information, such as SNPs, in applied medicine.

In general, 10%-to-15% of SNPs will affect protein function by alteringspecific amino acid residues, will affect the proper processing of genesby changing splicing mechanisms, or will affect the normal level ofexpression of the gene or protein by varying regulatory mechanisms. Itis envisioned that the identification of informative SNPs will lead tomore accurate diagnosis of inherited disease, better prognosis of risksusceptibilities, or identity of sporadic mutations in tissue. Oneapplication of an individual's SNP profile would be to significantlydelay the onset or progression of disease with prophylactic drugtherapies. Moreover, an SNP profile of drug metabolizing genes could beused to prescribe a specific drug regimen to provide safer and moreefficacious results. To accomplish these ambitious goals, genomesequencing will move into the resequencing phase with the potential ofpartial sequencing of a large majority of the population, which wouldinvolve sequencing specific regions or single base pairs in parallel,which are distributed throughout the human genome to obtain the SNPprofile for a given complex disease.

Sequence variations underlying most common diseases are likely toinvolve multiple SNPs, which are dispersed throughout associated genesand exist in low frequency. Thus, DNA sequencing technologies thatemploy strategies for de novo sequencing are more likely to detectand/or discover these rare, widely dispersed variants than technologiestargeting only known SNPs.

Traditionally, DNA sequencing has been accomplished by the “Sanger” or“dideoxy” method, which involves the chain termination of DNA synthesisby the incorporation of 2′,3′-dideoxynucleotides (ddNTPs) using DNApolymerase (Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNAsequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74, 5463-5467). The reaction also includes the natural2′-deoxynucleotides (dNTPs), which extend the DNA chain by DNAsynthesis. Balanced appropriately, competition between chain extensionand chain termination results in the generation of a set of nested DNAfragments, which are uniformly distributed over thousands of bases anddiffer in size as base pair increments. Electrophoresis is used toresolve the nested DNA fragments by their respective size. The ratio ofdNTP/ddNTP in the sequencing reaction determines the frequency of chaintermination, and hence the distribution of lengths of terminated chains.The fragments are then detected via the prior attachment of fourdifferent fluorophores to the four bases of DNA (i.e., A, C, G, and T),which fluoresce their respective colors when irradiated with a suitablelaser source. Currently, Sanger sequencing has been the most widely usedmethod for discovery of SNPs by direct PCR sequencing (Gibbs, R. A.,Nguyen, P.-N., McBride, L. J., Koepf, S. M., and Caskey, C. T. (1989)Identification of mutations leading to the Lesch-Nyhan syndrome byautomated direct DNA sequencing of in vitro amplified cDNA. Proc. Natl.Acad. Sci. USA 86, 1919-1923) or genomic sequencing (Hunkapiller, T.,Kaiser, R. J., Koop, B. F., and Hood, L. (1991) Large-scale andautomated DNA sequencing Determination. Science 254, 59-67;International Human Genome Sequencing Consortium. Initial sequencing andanalysis of the human genome. (2001) Nature 409, 860-921).

Another promising sequencing approach is cyclic reversible termination(CRT), which is a cyclic method of detecting the synchronistic, singlebase additions of multiple templates. This approach differentiatesitself from the Sanger method (Metzker, M. L. (2005) Genome Res. 15,1767-1776) in that it can be performed without the need for gelelectrophoresis, a major bottleneck in advancing this field. Like Sangersequencing, however, longer read-lengths translates into fewersequencing assays needed to cover the entire genome.

It has remained difficult to accomplish the goal of long CRT readsbecause reversible terminators typically act as poor substrates withcommercially available DNA polymerases. Reversible terminators arestructured with a 3′-O-blocking group and a nucleobase attachedfluorescent dye via a linking group. Both blocking and dye groupsrequire removal prior to subsequent base additions. These nucleotidemodifications are not well tolerated by DNA polymerases, which can bemutated by numerous strategies to improve enzymatic performance. Upondeprotection, the nucleobase linker group is left behind, accumulatingsequentially in the growing DNA duplex with subsequent CRT cycles. It isbelieved that poor enzyme kinetics and a sequentially modified DNAduplex limit longer read-lengths. The present invention describes novel,reversible nucleotide structures that require a single attachment ofboth terminating and fluorescent dye moieties, improving enzyme kineticsas well as deprotection efficiencies. These reversible terminators areincorporated efficiently by a number of commercially available DNApolymerases, with the deprotection step transforming the growing DNAduplex into its natural state.

DNA sequencing read-lengths of CRT technologies are governed by theoverall efficiency of each nucleotide addition cycle. For example, ifone considers the end-point of 50% of the original starting material ashaving an acceptable signal-to-noise ratio, the following equation canbe applied to estimate the effect of the cycle's efficiency onread-length: (RL)^(Ceff)=0.5, where RL is the read-length in bases andC_(eff) is the overall cycle efficiency. In other words, a read-lengthof 7 bases could be achieved with an overall cycle efficiency of 90%, 70bases could be achieved with a cycle efficiency of 99% and 700 baseswith a cycle efficiency of 99.9%. To achieve the goal of sequencinglarge stretches, the method must provide very high cycle efficiency orthe recovery may fall below acceptable signal to noise ratios.Reversible terminators that exhibit higher incorporation anddeprotection efficiencies will achieve higher cycle efficiencies, andthus longer read-lengths.

For CRT terminators to function properly, the protecting group must beefficiently cleaved under mild conditions. The removal of a protectinggroup generally involves either treatment with strong acid or base,catalytic or chemical reduction, or a combination of these methods.These conditions may be reactive to the DNA polymerase, nucleotides,oligonucleotide-primed template, or the solid support creatingundesirable outcomes. The use of photochemical protecting groups is anattractive alternative to rigorous chemical treatment and can beemployed in a non-invasive manner.

A number of photoremovable protecting groups including 2-nitrobenzyl,benzyloxycarbonyl, 3-nitrophenyl, phenacyl, 3,5-dimethoxybenzoinyl,2,4-dinitrobenzenesulphenyl, and their respective derivatives have beenused for the syntheses of peptides, polysaccharides, and nucleotides(Pillai, V. N. R. (1980) Photoremovable Protecting Groups in OrganicSynthesis. Synthesis, 1-26). Of these, the light sensitive 2-nitrobenzylprotecting group has been successfully applied to the 2′-OH ofribonucleosides for diribonucleoside synthesis (Ohtsuka, E., Tanaka, S.,and Ikehara, M. (1974) Studies on transfer ribonucleic acids and relatedcompounds. IX(1) Ribooligonucleotide synthesis using a photosensitiveo-nitrobenzyl protection at the 2′-hydroxyl group. Nucleic Acids Res. 1,1351-1357), the 2′-OH of ribophosphoramidites in automated ribozymesynthesis (Chaulk, S. G., and MacMillan, A. M. (1998) Caged RNA:photo-control of a ribozyme. Nucleic Acids Res. 26, 3173-3178), the3′-OH of phosphoramidites for oligonucleotide synthesis in theAffymetrix chemistry (Pease, A. C., Solas, D., Sullivan, E. J., Cronin,M. T., Holmes, C. P., and Fodor, S. P. A. (1994) Light-generatedoligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl.Acad. Sci. USA 91, 5022-5026), and to the 3′-OH group for DNA sequencingapplications (Metzker, M. L., Raghavachari, R., Richards, S., Jacutin,S. E., Civitello, A., Burgess, K., and Gibbs, R. A. (1994) Terminationof DNA synthesis by novel 3′-modified deoxyribonucleoside triphosphates.Nucleic Acids Res. 22, 4259-4267). Under deprotection conditions(ultraviolet light >300 nm), the 2-nitrobenzyl group can be efficientlycleaved without affecting either the pyrimidine or purine bases (Pease,A. C., Solas, D., Sullivan, E. J., Cronin, M. T., Holmes, C. P., andFodor, S. P. A. (1994) Light-generated oligonucleotide arrays for rapidDNA sequence analysis. Proc. Natl. Acad. Sci. USA 91, 5022-5026) and(Bartholomew, D. G., and Broom, A. D. (1975) One-step Chemical Synthesisof Ribonucleosides bearing a Photolabile Ether Protecting Group. J.Chem. Soc. Chem. Commun., 38).

The need for developing new sequencing technologies has never beengreater than today with applications spanning diverse research sectorsincluding comparative genomics and evolution, forensics, epidemiology,and applied medicine for diagnostics and therapeutics. Currentsequencing technologies are too expensive, labor intensive, and timeconsuming for broad application in human sequence variation studies.Genome center cost is calculated on the basis of dollars per 1,000 Q₂₀bases and can be generally divided into the categories ofinstrumentation, personnel, reagents and materials, and overheadexpenses. Currently, these centers are operating at less than one dollarper 1,000 Q₂₀ bases with at least 50% of the cost resulting from DNAsequencing instrumentation alone. Developments in novel detectionmethods, miniaturization in instrumentation, microfluidic separationtechnologies, and an increase in the number of assays per run will mostlikely have the biggest impact on reducing cost.

It is therefore an object of the invention to provide novel compoundsthat are useful in efficient sequencing of genomic information in highthroughput sequencing reactions.

It is another object of the invention to provide novel reagents andcombinations of reagents that can efficiently and affordably providegenomic information.

It is yet another object of the invention to provide libraries andarrays of reagents for diagnostic methods and for developing targetedtherapeutics for individuals.

SUMMARY

Provided are nucleoside compounds as well as phosphates and saltsthereof, that can be used in DNA sequencing technology. The compoundsare optionally in the form of ribonucleoside triphosphate (NTP) anddeoxyribonucleoside triphosphate (dNTP) compounds. The nucleotide andnucleoside compounds include a photocleavable group lableled with afluorescent dye. The nucleotide and nucleoside compounds containingphotocleavable protecting groups are designed to terminate DNA synthesisand then be cleave efficiently, so that nucleic acid oligomers can besequenced rapidly in a parallel format. The presence of such cleavablegroups lableled with fluorescent dyes on the nucleotide and nucleosidecompounds can enhance the speed and accuracy of sequencing of largeoligomers of DNA in parallel, to allow, for example, rapid whole genomesequencing, and the identification of polymorphisms and other valuablegenetic information.

A variety of nucleotide and nucleoside compounds, containing thenucleobases adenine, cytosine, guanine, thymine, uracil, or naturallyoccurring derivatives thereof, are provided that include cleavablegroups and/or which can be derivatized to include a detectable labelsuch as a dye.

In one embodiment the base of the nucleoside covalently attached with a2-nitrobenzyl group, and the alpha carbon position of the 2-nitrobenzylgroup is optionally substituted with one alkyl or aryl group asdescribed herein. The 2-nitrobenzyl group can be functionalized toenhance the termination properties as well as the light catalyzeddeprotection rate. The termination properties of the 2-nitrobenzyl andalpha carbon substituted 2-nitrobenzyl group attached to the nucleobaseoccur even when the 3′-OH group on the ribose sugar is unblocked. These3′-OH unblocked terminators are well-tolerated by a number ofcommercially available DNA polymerases, representing a key advantageover 3′-O-blocked terminators. The alpha carbon substituted2-nitrobenzyl group also can be derivatized to include a selectedfluorescent dye.

In one embodiment the base of the nucleoside is covalently attached witha 2-nitrobenzyl group, and the 2-nitrobenzyl group is optionallysubstituted with one or more of an electron donating and electronwithdrawing group as described herein. The 2-nitrobenzyl group can befunctionalized to enhance the light catalyzed deprotection rate. The2-nitrobenzyl group also can be derivatized to include a detectablefluorescent dye.

In particular, methods for DNA sequencing are provided usingcombinations of the four nucleoside triphosphate compounds, modifiedwith 2-nitrobenzyl groups, and derivatives described herein and labeledwith distinct fluorescent dyes, which could be used for identifying theincorporated bases to reveal the underlying DNA sequence.

DETAILED DESCRIPTION

Provided are nucleotide and nucleoside compounds as well as salts,esters and phosphates thereof, that can be used in rapid DNA sequencingtechnology. The compounds are optionally in the form of ribonucleosidetriphosphates (NTPs) and deoxyribonucleoside triphosphates (dNTP). Thenucleotide and nucleoside compounds in one embodiment includes aphotocleavable group labeled with a fluorescent dye. The nucleotide andnucleoside compounds include photoremovable protecting groups that aredesigned to terminate DNA synthesis as well as cleave rapidly, so thatthese monomers can be used for rapid sequencing in a parallel format.The presence of such rapidly cleavable groups labeled with fluorescentdyes on the nucleotide and nucleoside compounds can enhance the speedand accuracy of sequencing of large oligomers of DNA in parallel, toallow, for example, rapid whole genome sequencing, and theidentification of polymorphisms and other valuable genetic information.

A variety of nucleotide and nucleoside compounds, containing thenucleobases adenine, cytosine, guanine, thymine, uracil, or naturallyoccurring derivatives thereof, are provided that include cleavablegroups and/or which can be derivatized to include a detectable labelsuch as a dye.

In one embodiment, the nucleobases adenine, cytosine, guanine, thymine,uracil, or naturally occurring derivatives thereof, can be covalentlyattached to a photoremovable protecting group such as a 2-nitrobenzylgroup. The 2-nitrobenzyl group can be derivatized to enhance itstermination of DNA synthesis as well as deprotection rate, thusincreasing its usefulness in DNA sequencing. The photoremovableprotecting group, such as the 2-nitrobenzyl group, also can bederivatized with a fluorescent dye by covalent linkage to thephotoremovable protecting group.

I. Advantages of Compounds for Sequencing

Nucleotide and nucleoside compounds are provided which are useful in DNAsequencing technology. Cyclic reversible termination (CRT) is a cyclicmethod of detecting the synchronous, single base additions of multipletemplates. This approach differentiates itself from the Sanger method inthat it can be performed without the need for gel electrophoresis andhighly-parallel format, which are major bottlenecks in advancing thisfield. Like Sanger sequencing, however, longer read-lengths translatesinto fewer sequencing assays needed to cover the entire genome.

The CRT cycle comprises three steps, incorporation, imaging, anddeprotection. For this procedure, cycle efficiency, cycle time, andsensitivity are important factors. The cycle efficiency is the productof deprotection and incorporation efficiencies and determines the CRTread-length. The CRT cycle time is the sum of incorporation, imaging,and deprotection times. For rapid CRT for whole genome sequencing, thenucleotide and nucleoside compounds as disclosed herein may be used,which can exhibit fast and efficient deprotection properties. Thesecompounds can be labeled with fluorescent dyes, attached directly to the2-nitrobenzyl, providing fluorescent, reversible terminators withsimilar deprotection properties.

The read-length of CRT technologies are governed by the overallefficiency of each nucleotide addition cycle, product of deprotectionand incorporation efficiencies. For example, if one considers theend-point of 50% of the original starting material as having anacceptable signal-to-noise ratio, the following equation can be appliedto estimate the effect of the cycle's efficiency on read-length:(RL)^(Ceff)=0.5where RL is the read-length in bases and Ceff is the overall cycleefficiency. In other words, a read-length of 7 bases could be achievedwith an overall cycle efficiency of 90%, 70 bases could be achieved witha cycle efficiency of 99% and 700 bases with a cycle efficiency of99.9%. The efficiency of incorporation of compounds according to theinvention may range from about 70% to about 100% of the incorporation ofthe analogous native nucleoside. Preferably, the efficiency ofincorporation will range from about 85% to about 100%. Photocleavageefficiencies will preferably range from about 85% to about 100%.Further, termination of nucleic acid extension will range from about 90%to about 100% upon incorporation of compounds according to theinvention. Nucleotide and nucleoside compounds in one embodiment have acycle efficiency of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,or 99.9%.

When applied to genomic DNA, the compounds can be used in CRT to readdirectly from genomic DNA. Fragmented genomic DNA can be hybridized to ahigh-density oligonucleotide chip containing priming sites that spanselected chromosomes. Each priming sequence is separated by theestimated read-length of the CRT method. Between base additions, afluorescent imager can simultaneously image the entire high-densitychip, marking significant improvements in speed and sensitivity. Thefluorophore, which is attached to the 2-nitrobenzyl group or itsderivatives described herein, is removed by UV irradiation releasing the2-nitrobenzyl group for the next round of base addition. Afterapproximately 500 CRT cycles, the complete and contiguous genomesequence information can then be compared to the reference human genometo determine the extent and type of sequence variation in anindividual's sample.

II. Compounds

A variety of nucleosides and compounds as well as their mono, di andtriphosphates are provided. The compounds are useful for DNA sequencingtechnology. In one embodiment, the nucleotide and nucleoside compoundsinclude a photocleavable terminating group labeled with a fluorescentdye that can be detected and efficiently cleaved in CRT reactions. Thenucleotide and nucleoside compounds can be converted into theirrespective natural nucleoside monophosphates for subsequent rounds ofDNA polymerase reactions.

In a particular embodiment, a nucleotide and nucleoside compounds areprovided comprising a deoxyribose or ribose sugar and a base, whereinthe base is covalently linked to a photocleavable terminating,2-nitrobenzyl group. The 2-nitrobenzyl group can be substituted withgroups that increase termination of DNA synthesis as well as the rate ofdeprotection. In addition, the 2-nitrobenzyl group can be detectable byattaching a reporter group, such as a dye. The dye may be optionallylinked to 2-nitrobenzyl group by a bifunctional linker. Compoundsaccording to the invention may be represented by the following formula:

wherein R₁ is H, monophosphate, diphosphate or triphosphate, R₂ is H orOH, base is cytosine, uracil, thymine, adenine, or guanine, or naturallyoccurring derivatives thereof, cleavable terminating moiety is a groupimparting polymerase termination properties to the compound, linker is abifunctional group, and dye is a fluorophore.

Compounds according to the invention can be designed as fluorescent,photolabile reversible terminators useful in DNA synthesis sequencing.The compounds can be optimized reversible terminators, modified to havefast and efficient deprotection behavior and good fluorescent propertiesin aqueous solutions. In one embodiment, a compound is provided having astructure of formulas I-VII:

wherein R₁═H, monophosphate, diphosphate or triphosphate, R₂═H or OH, R₃and R₄ are each independently selected from the group of H, a C₁-C₁₂straight chain or branched alkyl, a C₂-C₁₂ straight chain or branchedalkenyl or polyenyl, a C₂-C₁₂ straight chain or branched alkynyl orpolyalkynyl, and an aromatic group such as a phenyl, naphthyl, orpyridine ring, with the proviso that at least one of R₃ and R₄ is H, R₅,R₆, R₇, and R₈ are each independently selected from the group H, OCH₃,NO₂, CN, a halide, a C₁-C₁₂ straight chain or branched alkyl, a C₂-C₁₂straight chain or branched alkenyl or polyenyl, a C₂-C₁₂ straight chainor branched alkynyl or polyalkynyl an aromatic group such as a phenyl,naphthyl, or pyridine ring, and/or a linker group of the generalstructure:

X═CH₂, CH═CH, C≡C, O, S, or NH, Y═CH₂, O, or NH, n=an integer from 0-12;m=an integer from 0-12, and Dye=a fluorophore, or pharmaceuticallyacceptable salt or ester thereof or enantiomer, racemic mixture, orstereoisomer thereof.

In a particular embodiment, in the compounds provided herein comprisinga derivatized 2-nitrophenyl ring, such as the compounds of formulasI-VII, the rate of deprotection and removal of the 2-nitrophenyl groupduring DNA sequencing can be enhanced by including an electron donatinggroup at the 4-position or an electron withdrawing group at the5-position of the 2-nitrophenyl ring.

In a preferred embodiment, R₃ and R₄ are selected from the groupconsisting of, but not limited to, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, isopropyl,tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl. Alternatively,R₃ and R₄ are selected from the group consisting of, but not limited to,alkyl and aromatic groups optionally containing at least one heteroatomin the alkyl or aromatic groups, and further wherein the aromatic groupmay optionally be an aryl such as phenyl or polycyclic such as anaphthyl group. In certain embodiments, R₅, R₆, R₇, and R₈ are selectedfrom an aromatic group consisting of aryl and polycyclic groups.

Alternatively, photocleavable terminating moieties may have thefollowing general structures:

For example, compounds with such photocleavable terminating moietiescould have the following structures:

A compound according to claim 1, wherein the cleavable terminatingmoiety is attached to the base through a linkage selected from the groupconsisting of benzyl amine, benzyl ether, carbamate, carbonate,2-(o-nitrophenyl)ethyl carbamate, and 2-(o-nitrophenyl)ethyl carbonate.Such embodiments are within the scope of the current invention.

Fluorescent dyes are not particularly limited. For example, theflurophore may be seclected from the group consisting of, but notlimited to, BODIPY, fluoroscein, rhodamine, coumarin, xanthene, cyanine,pyrene, phthalocyanine, phycobiliprotein, ALEXA FLUOR® (e.g., ALEXAFLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXAFLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXAFLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXAFLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXAFLUOR® 700 or ALEXA FLUOR® 750),squariane dye, combinations resulting inenergy transfer dyes, and derivatives thereof.

Preferred embodiments include but are not limited to the followingcompounds:

III. Synthesis of Compounds

The compounds disclosed herein can be synthesized generally as disclosedherein, and using methods available in the art. For example, thefollowing general scheme represents the synthesis of an adenosinecompound:

Additional details are provided in the Examples section.IV. Methods of Use of Compounds According to the Invention

The nucleoside compounds disclosed herein can be used in for a varietyof purposes in DNA sequencing technology. Polymerases used inconjunction with the compounds according to the invention may be nativepolymerases or modified polymerases. Polymerases include withoutlimitation Taq DNA polymerase, Klenow(-exo-) DNA polymerase, Bst DNApolymerase, VENT® (exo-) DNA polymerase (DNA polymerase A cloned fromThermococcus litoralis and containing the D141A and E143A mutations),Pfu(-exo-) DNA polymerase, and DEEPVENT™ (exo-) DNA polymerase (DNApolymerase A, cloned from the Pyrococcus species GB-D, and containingthe D141A and E143A mutations). Modified polymerases include withoutlimitation AMPLITAQ® DNA polymerase, FS (Taq DNA polymerase thatcontains the G46D and F667Y mutations), THERMOSEQUENASE™ DNA polymerase(Taq DNA polymerase that contains the F667Y mutation), THERMOSEQUENASE™II DNA polymerase (blend of THERMOSEQUENASE™ DNA polymerase and T.acidophilum pyrophosphatase), THERMINATOR™ DNA polymerase (DNApolymerase A, cloned from the Thermococcus species 9° N-7 and containingthe D141A, E143A and A485L mutations), THERMINATOR™ II DNA polymerase(THERMINATOR™ DNA polymerase that contains the additional Y409Vmutation), and VENT® (exo-) A488L DNA polymerase (VENT® (exo-) DNApolymerase that contains the A488L mutation). Preferably, compoundsaccording to the invention are incorporated at levels equal to or nearthe incorporation levels of naturally-occurring nucleotides, thusresulting in no bias against the compounds according to the invention.Even more preferably, compounds according to the invention arecompatible with commercially-available polymerases.

In one embodiment, the compounds can be used in cyclic reversibletermination (CRT), which is a cyclic method of detecting thesynchronous, single base additions of multiple templates. Longerread-lengths translate into fewer sequencing assays needed to cover theentire genome. A method of synthesizing a nucleic acid comprises thefollowing steps: attaching the 5′-end of a primer to a solid surface;hybridizing a target nucleic acid to the primer attached to the solidsurface; adding one or more compounds according to the formula

wherein R₁ is H, monophosphate, diphosphate or triphosphate, R₂ is H orOH, base is cytosine, uracil, thymine, adenine, or guanine, or naturallyoccurring derivatives thereof, cleavable terminating moiety is a groupimparting polymerase termination properties to the compound, linker is abifunctional group, and dye is a fluorophore; adding DNA polymerase tothe hybridized primer/target nucleic acid complex to incorporate thecompound of the previous step into the growing primer strand, whereinthe incorporated compound terminates the polymerase reaction at anefficiency of between about 90% to about 100%; washing the solid surfaceto remove unincorporated components; detecting the incorporatedfluorophore, wherein the detector is optionally a pulsed multilineexcitation detector for imaging fluorescent dyes; optionally adding oneor more chemical compounds to permanently cap unextended primers;exposing the solid support to a light source to remove thephotocleavable moiety resulting in an extended primer withnaturally-occurring components; washing the solid surface to remove thecleaved protecting group; and repeating the above steps in a cyclicfashion.

In another embodiment, compounds according to the invention can be usedin a method of determining the sequence of a nucleic acid moleculecomprising the steps of adding a target nucleic acid molecule to asequencing apparatus, adding one or more compounds according to theinvention to the sequencing apparatus, adding a polymerase enzyme andoptionally naturally-occurring nucleic acid components to the sequencingapparatus, performing a polymerase reaction to incorporate at least oneof the compounds of the previous step into a growing nucleic acidstrand, and analyzing the result of the polymerase reaction forincorporation of at least one compound according to the invention. Thesteps can be performed in any order and in any number of iterations.Preferably, the incorporation step is followed by termination of strandgrowth at a rate of from about 90% to about 100%. In another embodiment,the incorporation of the inventive compound occurs at about 70% to about100% of the rate of incorporation of a native substrate of the analogousbase, such that no significant bias occurs. For example, theincorporation rate may occur at about 85% to about 100% of the normalrate for the corresponding nucleotide base. An important embodimentincludes the step of exposing the nucleic acid molecule resulting fromincorporation of a modified nucleotide to a UV or light source to removea photocleavable terminating moiety from the nucleic acid. Preferably,the efficiency of the photocleavage step is about 85% to about 100% fromexposure to the UV or light source.

Methods according to the invention can be practiced individually or incombination. For example, the method of sequencing a nucleic acidmolecule can be practiced in part as a method of incorporating anon-naturally occurring component into a nucleic acid molecule, or aseparate method of converting a non-naturally occurring component in anucleic acid molecule into a naturally-occurring component followingincorporation of a compound according to the invention.

In one embodiment, methods according to the invention include the aspectof terminating nucleic acid synthesis following incorporation of anunprotected 3′-OH nucleotide. Advantageously, an unprotected 3′-OHnucleotide can be incorporated by polymerases at a higher level,resulting in lower levels of modified nucleotide present in thepolymerase reaction and lower bias compared to natural nucleotides.Therefore, a preferred embodiment includes a method of terminatingnucleic acid synthesis comprising the step of placing a 3′-OHunprotected nucleotide or nucleoside in the environment of a polymeraseand allowing incorporation of the 3′-OH unprotected nucleotide ornucleoside into a nucleic acid molecule, wherein the 3′-OH unprotectednucleotide or nucleoside is a compound according to the followingformula:

wherein R₁ is H, monophosphate, diphosphate or triphosphate, R₂ is H orOH, base is cytosine, uracil, thymine, adenine, or guanine, or naturallyoccurring derivatives thereof, cleavable terminating moiety is a groupimparting polymerase termination properties to the compound, linker is abifunctional group, and dye is a fluorophore. Preferrably, the methodhas an efficiency of termination upon incorporation of the 3′-OHunprotected nucleotide or nucleoside ranging from about 90% to about100%. Alternatively, the method may have an efficiency of incorporationof the 3′-OH unprotected nucleotide or nucleoside ranges from about 70%to about 100% compared to the efficiency of incorporation of anaturally-occurring nucleotide or nucleoside with the same base.

The nucleotide and nucleoside compounds can be used in CRT to readdirectly from genomic DNA. Fragmented genomic DNA can be hybridized to ahigh-density oligonucleotide chip containing priming sites that spanselected chromosomes. Each priming sequence is separated by theestimated read-length of the CRT method. Between base additions, afluorescent imager, such as a Pulsed Multiline Excitation (PME)detector, can simultaneously image the entire high-density chip, markingsignificant improvements in speed and sensitivity. The fluorophore,which is attached to the cleavable terminating group on the base, isremoved by UV irradiation transforming the modified nucleotide into itsnatural from for the next round of base addition. After approximately500 CRT cycles, the completed and contiguous genome sequence informationcan then be compared to the reference human genome to determine theextent and type of sequence variation in an individual's sample. Methodsfor cyclic reversible termination have been developed in the art and canbe used, as described, e.g., in WO 2003/021212, the disclosure of whichis incorporated herein by reference.

In one embodiment, a method for sequencing a nucleic acid by detectingthe identity of a nucleotide analogue after the nucleotide analogue isincorporated into a growing strand of DNA in a polymerase reaction isused, which comprises the following steps:

-   -   (a) attaching the 5′ end of a nucleic acid to a solid surface;    -   (b) attaching a primer to the nucleic acid attached to the solid        surface;    -   (c) adding a polymerase and one or more different nucleoside        triphosphate compounds to the nucleic acid wherein the        nucleoside triphosphate compound incorporates and then        terminates the polymerase reaction and wherein each nucleoside        triphosphate compound comprises a base selected from the group        consisting of adenine, guanine, cytosine, thymine, and uracil,        and their analogues and a photocleavable terminating group        attached to the base, the photocleavable group comprising a        detectable label, and a deoxyribose or ribose sugar,    -   (d) optionally washing the solid surface to remove        unincorporated nucleotide analogues;    -   (e) detecting and thereby identifying the detectable label, such        as a fluorescent dye or reporter molecule, attached to the        terminated nucleoside triphosphate, e.g. with PME;    -   (f) optionally adding one or more chemical compounds to        permanently cap any unreacted —OH group on the primer attached        to the nucleic acid or on a primer extension strand formed by        adding one or more nucleotides to the primer;    -   (g) exposing the solid surface to a light source to remove the        photocleavable protecting group containing the unique label or        reporter molecule, wherein the remaining incorporated nucleoside        monophosphate unit resembles a natural, native, or unmodified        nucleic acid molecule;    -   (h) washing the solid surface to remove the cleaved protecting        group; and    -   (i) repeating steps (c) through (h) for determining the sequence        of identified incorporated nucleotide analogs into the growing        primer strand.

PME Detector

In one embodiment, a pulsed-multiline excitation (“PME”) for color-blindfluorescence detection can be used as described in US 2003/0058440published Mar. 27, 2003, or WO 2003/021212, published Mar. 13, 2003.This technology provides fluorescence detection with application forhigh-throughput identification of informative SNPs, for more accuratediagnosis of inherited disease, better prognosis of risksusceptibilities, or identification of sporadic mutations. The PMEtechnology has two main advantages that significantly increasefluorescence sensitivity: (1) optimal excitation of all fluorophores inthe genomic assay and (2) “color-blind” detection, which collectsconsiderably more light than standard wavelength resolved detection.This technology differs significantly from DNA sequencinginstrumentation which features single source excitation and colordispersion for DNA sequence identification. The technology can be usedin clinical diagnostics, forensics, and general sequencing methodologiesand will have the capability, flexibility, and portability of targetedsequence variation assays for a large majority of the population.

In one embodiment, an apparatus and method for use in high-throughputDNA sequence identification is used. A pulse-multiline excitationapparatus for analyzing a sample containing one or more fluorescentspecies is used, comprising: one or more lasers configured to emit twoor more excitation lines, each excitation line having a differentwavelength; a timing circuit coupled to the one or more lasers andconfigured to generate the two or more excitation lines sequentiallyaccording to a timing program to produce time-correlated fluorescenceemission signals from the sample; a non-dispersive detector positionedto collect the time-correlated fluorescence emission signals emanatingfrom the sample; and an analyzer coupled to the detector and configuredto associate the time-correlated fluorescence emission signals with thetiming program to identify constituents of the sample.

The detector and the analyzer may be integral. In one embodiment, thetwo or more excitation lines intersect at the sample, or the two or moreexcitation lines may be configured so that they do not intersect in thesample. The two or more excitation lines may be coaxial.

In one embodiment, the apparatus may further comprise an assembly of oneor more prisms in operative relation with the one or more lasers andconfigured to render radiation of the two or more excitation linessubstantially colinear and/or coaxial.

The apparatus may have a plurality of excitation lines, for example 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more excitationlines having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ormore excitation wavelengths, respectively. The sample may be comprised aplurality of vessels such as capillaries, for example in 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, up to 20, up to 24, up to 28, upto 36, up to 48, up to 64, up to 96, up to 384 or more capillaries. Asheath flow cuvette may be used.

The timing program may comprise a delay between the firing of each laserof between about 10 fs and about 5 s, between about 1 ms and about 100ms, or between about 50 ps and about 500 ps. One or more of theexcitation lines is pulsed. The pulsed excitation line may be controlledby TTL logic or by mechanical or electronic means. In one embodiment,the apparatus may generate a sequence of discrete excitation lines thatare time-correlated with the fluorescence emission signals from thesample.

The lasers may independently comprise a diode laser, a semiconductorlaser, a gas laser, such as an argon ion, krypton, or helium-neon laser,a diode laser, a solid-state laser such as a Neodymium laser which willinclude an ion-gain medium, such as YAG and yttrium vanadate (YVO₄), ora diode pumped solid state laser. Other devices, which produce light atone or more discrete excitation wavelengths, may also be used in placeof the laser. The laser may further comprise a Raman shifter in operablerelation with at least one laser beam. In one embodiment of theinvention, the excitation wavelength provided by each laser is opticallymatched to the absorption wavelength of each fluorophore.

The detector may comprise a charged couple device, a photomultipliertube, a silicon avalanche photodiode or a silicon PIN detector. Thefootprint of the device is preferably small, such as less than 4 ft×4ft×2 ft, less than 1 ft×1 ft×2 ft, and could be made as small as 1 in×3in×6 in.

Another aspect comprises a method of identifying sample componentscomprising: (a) preparing a sample comprising sample components, a firstdye and a second dye; (b) placing the sample in the beam path of a firstexcitation line and a second excitation line; (c) sequentially firingthe first excitation line and the second excitation line; (d) collectingfluorescence signals from the samples as a function of time; and (e)sorting the fluorescence by each excitation line's on-time window,wherein the sample components are identified. It is an aspect of theinvention that the fluorescence signals are collected from discrete timeperiods in which no excitation line is incident on the sample, the timeperiods occurring between the firing of the two excitation lines. Thistechnique is known as “looking in the dark.” Yet another aspect is thatthe absorption maximum of the first dye substantially corresponds to theexcitation wavelength of the first excitation line. The absorptionmaximum of the second dye may also substantially corresponds to theexcitation wavelength of the second excitation line. In yet anotheraspect there is a third and fourth dye and a third and fourth excitationline, wherein the absorption maxima of the third and fourth dyessubstantially correspond to the excitation wavelengths of the third andfour excitation lines, respectively. Similarly, there may be 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16 or more dyes wherein the absorption maximaof the dyes substantially corresponds to excitation wavelengths of a5th, 6th, 7th, 8th, 9th, 10th, 11 th, 12th, 13th, 14th, or moreexcitation lines, respectively. The dyes may be a zanthene, fluorescein,rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine,phycobiliprotein, ALEXA FLUOR® (e.g., ALEXA FLUOR® 350, ALEXA FLUOR®405, ALEXA FLUOR® 430, ALEXA FLUOR® 488, ALEXA FLUOR® 514, ALEXA FLUOR®532, ALEXA FLUOR® 546, ALEXA FLUOR® 555, ALEXA FLUOR® 568, ALEXA FLUOR®568, ALEXA FLUOR® 594, ALEXA FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR®647, ALEXA FLUOR® 660, ALEXA FLUOR® 680, ALEXA FLUOR® 700 or ALEXAFLUOR® 750), squariane dyes, or some other suitable dye.

In one embodiment, sample components enable the determination of SNPs.The method may be for the high-throughput identification of informativeSNPs. The SNPs may be obtained directly from genomic DNA material, fromPCR amplified material, or from cloned DNA material and may be assayedusing a single nucleotide primer extension method. The single nucleotideprimer extension method may comprise using single unlabeled dNTPs,single labeled dNTPs, single 3′-modified dNTPs, single base-modified2′-dNTPs, single alpha-thio-dNTPs or single labeled 2′,3′dideoxynucleotides. The mini-sequencing method may comprise usingsingle unlabeled dNTPs, single labeled dNTPs, single 3′-modified dNTPs,single base-modified 2′-dNTPs, single alpha-thio-dNTPs or single labeled2′, 3′-dideoxynucleotides. The SNPs may be obtained directly fromgenomic DNA material, from PCR amplified material, or from cloned DNAmaterials.

Also envisioned are methods for detecting nucleic acids. Nucleic acidsmay be detected in situ or in various gels, blots, and similar methodsfor detecting nucleic acids, such as disclosed in U.S. Pat. No.7,125,660, which is incorporated herein by reference.

EXAMPLES Example 1 dA Compounds Synthesis of3′-O-(2-nitrobenzyl)-2′-deoxyadenosine triphosphate (WW1p108)

Scheme. Synthesis of3′-O-(2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate. (i) TBSCl,imidazole, DMF, room temperature, overnight; Boc₂O, DMAP, DMF, roomtemperature, overnight, 83%; (ii) n-Bu₄NF, THF, 0° C., then graduallywarmed to room temperature, 96%; (iii) TBSCl, imidazole, DMF, 83%; (iv)n-Bu₄NOH, NaI, NaOH, CH₂Cl₂/H₂O, 2-nitrobenzyl bromide, roomtemperature, 91%; (v) SiO₂, high vacuum, 70-80° C., 24 hours, 91%; (vi)n-Bu₄NF, THF, 0° C., then gradually warmed to room temperature, 23%;(vii) POCl₃, (MeO)₃PO, minus 20° C.; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 MHNEt₃HCO₃, 31%.

N⁶,N⁶-Bis-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.01)

Compound dA.01 was synthesized according to the procedure described byFurrer and Giese¹. A solution of 2′-deoxyadenosine dA (2.5 g, 10 mmol),imidazole (4.5 g, 66 mmol), and TBSCl (4.82 g, 32 mmol) in anhydrous DMF(25 mL) was stirred at room temperature overnight. Methanol (20 mL) wasadded, and the mixture was stirred for 20 minutes and then concentratedin vacuo. The residue was dissolved in anhydrous DMF (15 mL) followed bythe addition of DMAP (3.66 g, 30 mmol) and Boc₂O (6.55 g, 30 mmol). Thereaction was stirred at room temperature overnight and then concentratedin vacuo. The residue was dissolved in CH₂Cl₂ (100 mL) and washed twicewith saturated NH₄Cl solution (50 mL each). The combined aqueous layerwas extracted with CH₂Cl₂ (50 mL). The combined organic layer was thendried over Na₂SO₄, concentrated in vacuo, and purified by silica gelcolumn chromatography to giveN⁶,N⁶-bis-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.01 (5.66 g, 83%) as a white foam. ¹ Furrer, E. and Giese, B. (2003)On the distance-independent hole transfer over long (A•T)_(n)-sequencesin DNA. Helvetica Chimica Acta, 86, 3623-3632.

N⁶,N⁶-Bis-tert-butyloxycarbonyl-2′-deoxyadenosine (dA.02)

A solution of Bu₄NF (7.85 g, 30 mmol) in THF (30 mL) was added to asolution of compound dA.01 (6.78 g, 10 mmol) in THF (30 mL) at 0° C. Thereaction mixture was gradually warmed to room temperature, stirred fortwo hours, and then concentrated in vacuo. The residue was dissolved inCH₂Cl₂ (100 mL) and washed twice with saturated NH₄Cl solution (100 mLeach). The organic layer was then dried over Na₂SO₄, concentrated invacuo, and purified by silica gel column chromatography to yieldN⁶,N⁶-bis-tert-butyloxycarbonyl-2′-deoxyadenosine dA.02 (4.34 g, 96%) asa white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.84 (s, 1 H, H-8), 8.20 (s, 1 H, H-2), 6.41(dd, 1 H, J=5.6 Hz and 9.2 Hz, H-1′), 4.77 (d, 1 H, H-4′), 4.21 (s, 1 H,H-3′), 3.98 (dd, 1 H, H-5′a), 3.80 (m, 1 H, H-5′b), 3.00 (m, 1 H,H-2′a), 2.36 (m, 1 H, H-2′b), 1.47 (s, 18 H, (CH₃)₃CO).

N⁶,N⁶-Bis-tert-butyloxycarbonyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.03)

A solution of TBSCl (1.88 g, 12.5 mmol) in anhydrous DMF (5 mL) wasadded to a solution of compound dA.02 (4.34 g, 9.6 mmol) and imidazole(1.3 g, 19.2 mmol) in anhydrous DMF (20 mL) at 0° C. The mixture wasgradually warmed to room temperature and stirred for two days. Water (50mL) was added, and the mixture was extracted three times with ethylacetate (40 mL each). The combined organic layer was washed withsaturated NH₄Cl solution (50 mL), dried over Na₂SO₄, and purified bysilica gel column chromatography to yieldN⁶,N⁶-bis-tert-butyloxycarbonyl-5′-O-tert-butyl-dimethylsilyl-2′-deoxyadenosinedA.03 (4.68 g, 83%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.84 (s, 1 H, H-8), 8.42 (s, 1 H, H-2), 6.57(t, 1 H, J=6.4 Hz, H-1′), 4.69 (m, 1 H, H-4′), 4.10 (m, 1 H, H-3′), 3.89(m, 2 H, H-5′a and H-5′b), 2.70 (m, 1 H, H-2′a), 2.58 (m, 1 H, H-2′b),1.44 (s, 18 H, (CH₃)₃CO), 0.91 (s, 9 H, (CH₃)₃CSi), 0.10 (s, 6 H,(CH₃)₂Si).

N⁶,N⁶-Bis-tert-butyloxycarbonyl-5′-O-tert-butyldimethylsilyl-3′-O-(2-nitrobenzyl)-2′-deoxyadenosine(dA.04)

A solution of compound dA.03 (1.13 g, 2 mmol) in CH₂Cl₂ (3 mL) was mixedwith a solution of n-Bu₄NOH (0.94 mL, 4 mmol, 55% aqueous solution) andNaI (20 mg, catalytic amount) in NaOH (1 M; 3 mL). To the mixture, asolution of 2-nitrobenzyl bromide (1.3 g, 6 mmol) in CH₂Cl₂ (2 mL) wasadded dropwise, and the reaction mixture was stirred at room temperaturefor two hours in the dark. The organic layer was separated, and theaqueous layer was extracted twice with CH₂Cl₂ (10 mL each). The combinedorganic layer was dried over Na₂SO₄, concentrated in vacuo, and purifiedby silica gel column chromatography to yieldN⁶,N⁶-bis-tert-butyloxycarbonyl-5′-O-tert-butyldimethylsilyl-3′-O-(2-nitrobenzyl)-2′-deoxyadenosinedA.04 (1.28 g, 91%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.85 (s, 1 H, H-8), 8.43 (s, 1 H, H-2), 8.10(dd, 1 H, Ph-H), 7.81 (d, 1 H, Ph-H), 7.70 (t, 1 H, Ph-H), 7.51 (t, 1 H,Ph-H), 6.57 (t, 1 H, J=6.8 Hz, H-1′), 4.98 (dd, 2 H, PhCH₂), 4.45 (m, 1H, H-4′), 4.33 (m, 1 H, H-3′), 3.90 (m, 2 H, H-5′a and H-5′b), 2.73 (m,2 H, H-2′a and H-2′b), 1.46 (s, 18 H, (CH₃)₃CO), 0.91 (s, 9 H,(CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si);

ToF-MS (ESI): For the molecular ion C₃₃H₄₉N₆O₉Si [M+H]⁺, the calculatedmass was 701.3330, and the observed mass was 701.3317.

5′-O-tert-Butyldimethylsilyl-3′-O-(2-nitrobenzyl)-2′-deoxyadenosine(dA.05)

Silica gel 60 (10 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for 24 hours) was added to a solution of compounddA.04 (1.28 g, 1.8 mmol) in CH₂Cl₂ (50 mL), and the mixture wasevaporated in vacuo to dryness. The residue obtained was heated to70-80° C. for two days under reduced pressure, washed three times withmethanol (50 mL each), and filtered using a buchi funnel. The combinedfiltrate was concentrated in vacuo and purified by silica gel columnchromatography to yield5′-O-tert-butyldimethylsilyl-3′-O-(2-nitrobenzyl)-2′-deoxyadenosinedA.05 (0.83 g, 91%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.34 (s, 1 H, H-8), 8.15 (s, 1 H, H-2), 8.09(d, 1 H, Ph-H), 7.81 (d, 1 H, Ph-H), 7.67 (t, 1 H, Ph-H), 7.47 (t, 1 H,Ph-H), 6.50 (t, 1 H, J=6.8 Hz, H-1′), 6.03 (bs, 2 H, 6-NH₂), 4.96 (dd, 2H, PhCH₂), 4.43 (m, 1 H, H-4′), 4.30 (m, 1 H, H-3′), 3.88 (m, 2 H, H-5′aand H-5′b), 2.71 (m, 2 H, H-2′a and H-2′b), 0.91 (s, 9 H, (CH₃)₃CSi),0.10 (s, 6 H, (CH₃)₂Si);

ToF-MS (ESI): For the molecular ion C₂₃H₃₃N₆O₅Si [M+H]⁺, the calculatedmass was 501.2282, and the observed mass was 501.1702.

3′-O-(2-Nitrobenzyl)-2′-deoxyadenosine (dA.06)

A solution of n-Bu₄NF (314 mg, 1.2 mmol) in THF (1.2 mL) was added to asolution of compound dA.05 (400 mg, 0.8 mmol) in THF (3 mL) at 0° C. Thereaction mixture was gradually warmed to room temperature and stirredfor four hours. Methanol (10 mL) was added to dissolve the precipitateformed during the reaction, followed by the addition of silica gel 60(1.5 g). The mixture was evaporated in vacuo to dryness, and the residuewas purified by silica gel column chromatography to yield3′-O-(2-nitrobenzyl)-2′-deoxyadenosine dA.06 (72 mg, 23%) as a whitefoam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.35 (s, 1 H, H-8), 8.13 (s, 1 H, H-2),8.06 (d, 1 H, Ph-H), 7.79 (m, 2 H, Ph-H), 7.60 (m, 1 H, Ph-H), 7.34 (bs,2 H, 6-NH₂, D₂O exchangeable), 6.33 (dd, 1 H, J=4.8 and 6.8 Hz, H-1′),5.40 (t, 1 H, D₂O exchangeable, 5′-OH)), 4.92 (s, 2 H, PhCH₂), 4.36 (m,1 H, H-4′), 4.12 (m, 1 H, H-3′), 3.59 (m, 2 H, H-5′a and H-5′b), 2.85(m, 1 H, H-2′a), 2.54 (m, 1 H, H-2′b);

ToF-MS (ESI): For the molecular ion C₁₇H₁₉N₆O₅ [M+H]⁺, the calculatedmass was 387.1417, and the observed mass was 387.1350.

3′-O-(2-Nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate (WW1p108)

POCl₃ (26 μL, 0.24 mmol) was added to a solution of compound dA.06 (72mg, 0.18 mmol) in trimethylphosphate (1 mL), and maintained at minus20-30° C. for 2.5 hours. A solution of bis-tri-n-butylammoniumpyrophosphate (427 mg, 0.9 mmol) and tri-n-butylamine (0.2 mL) inanhydrous DMF (2 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to give3′-O-(2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate WW1p108 (38 mg,31%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.50 (s, 1 H, H-8), 8.24 (s, 1 H, H-2), 8.10(d, 1 H, Ph-H), 7.78 (d, 1 H, Ph-H), 7.62 (m, 1 H, Ph-H), 6.50 (dd, 1 H,J=6.8 and 8 Hz, H-1′), 5.03 (dd, 2 H, Ph-CH₂), 4.65 (m, 1 H, H-4′), 4.53(m, 1 H, H-3′), 4.22 (m, 2 H, H-5′a and H-5′b), 2.80 (m, 2 H, H-2′a andH-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.54 (d, J=19.4 Hz), −10.85 (d, J=19.4 Hz),−21.31 (t, J=19.4 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₁N₆O₁₄P₃Na [M+Na]⁺, thecalculated mass was 649.0226, and the observed mass was 649.0212.

The triphosphate was further purified using preparative HPLC without UVdetection to give sample free from contamination of the naturalnucleotide (e.g., dATP). Determination of the concentration of thetriphosphate solution was performed by UV/VIS measurement using theextinction coefficient of ε₂₆₀=20,800.

Synthesis of N⁶-(2-nitrobenzyl)-2′-deoxyadenosine triphosphate (WW1p129)

Scheme. Synthesis ofN⁶-(2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate. (i) Mg(ClO₄)₂,THF, 50° C., 68%; (ii) NaH, DMF, 2-nitrobenzyl bromide, 0° C., thengradually warmed to room temperature, 49%; (iii) SiO₂, vacuum, 70-80°C., 87%; (iv) n-Bu₄NF, THF, 43%; (v) POCl₃, (MeO)₃PO, minus 20-30° C.;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; 60%.

N⁶-tert-Butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.07)

Mg(ClO₄)₂ (155 mg, 0.7 mmol) was added to a solution of compound dA.01(2.36 g, 3.5 mmol) in anhydrous THF (35 mL) and stirred at 50° C.overnight. Solvent was removed in vacuo, and the crude product waspurified by silica gel column chromatography to giveN⁶-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.07 (1.39 g, 68%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.74 (s, 1 H, H-8), 8.29 (s, 1 H, H-2), 8.24(s, 1 H, 6-NHBoc), 6.49 (t, 1 H, J=6.4 Hz, H-1′), 4.60 (m, 1 H, H-4′),4.02 (m, 1 H, H-3′), 3.86 (dd, 1 H, H-5′a), 3.77 (dd, 1 H, H-5′b), 2.62(m, 1 H, H-2′a), 2.47 (m, 1 H, H-2′b), 1.54 (s, 9 H, (CH₃)₃CO), 0.90 (s,18 H, (CH₃)₃CSi), 0.08 (2 s, 12 H, (CH₃)₂Si).

N⁶-tert-Butyloxycarbonyl-N⁶-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.08)

NaH (5.3 mg, 0.22 mmol, dry) was added to a solution of compound dA.07(116 mg, 0.2 mmol) in anhydrous DMF (2 mL) at 0° C. and stirred for 30minutes. A solution of 2-nitrobenzyl bromide (43 mg, 0.2 mmol) inanhydrous DMF (0.5 mL) was added dropwise. The mixture was graduallywarmed to room temperature and stirred for two hours. DMF was removed invacuo, and the residue was dissolved in ethyl acetate (20 mL), washedtwice with saturated NH₄Cl solution (10 mL each) and once with water (10mL). The combined aqueous layer was extracted with ethyl acetate (10mL), and the combined organic layer was dried over Na₂SO₄, concentratedin vacuo, and purified by silica gel column chromatography to yieldN⁶-tert-butyloxycarbonyl-N⁶-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.08 (70 mg, 49%) as a viscous oil.

¹H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1 H, H-8), 8.38 (s, 1 H, H-2), 8.05(dd, 1 H, Ph-H), 7.77 (d, 1 H, Ph-H), 7.56 (m, 1 H, Ph-H), 7.40 (m, 1 H,Ph-H), 6.51 (t, 1 H, J=6.4 Hz, H-1′), 5.63 (s, 2 H, Ph-CH₂), 4.63 (m, 1H, H-4′), 4.03 (m, 1 H, H-3′), 3.87 (m, 1 H, H-5′a), 3.78 (m, 1 H,H-5′b), 2.63 (m, 1 H, H-2′a), 2.46 (m, 1 H, H-2′b), 1.40 (s, 9 H,(CH₃)₃CO), 0.92 (s, 18 H, (CH₃)₃CSi), 0.10 (2 s, 12 H, (CH₃)₂Si—);

ToF-MS (ESI): For the molecular ion C₃₄H₅₅N₆O₇Si₂ [M+H]⁺, the calculatedmass was 715.3671, and the observed mass was 715.3661.

N⁶-(2-Nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.09)

Silica gel 60 (3.5 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for 24 hours) was added to a solution of compounddA.08 (325 mg, 0.45 mmol) in CH₂Cl₂ (20 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 70-80° C.under reduced pressure for two days, washed three times with methanol(20 mL each), and filtered using a buchi funnel. The combined filtratewas concentrated in vacuo and purified by silica gel columnchromatography to yieldN⁶-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.09 (238 mg, 86%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1 H, H-8), 8.09 (s, 1 H, H-2), 8.07(d, 1 H, Ph-H), 7.74 (d, 1 H, Ph-H), 7.56 (m, 1 H, Ph-H), 7.42 (m, 1 H,Ph-H), 6.57 (t, 1 H, 6-NH), 6.44 (t, 1 H, J=6.4 Hz, H-1′), 5.19 (bs, 2H, Ph-CH₂), 4.61 (m, 1 H, H-4′), 4.00 (m, 1H, H-3′), 3.86 (dd, 1 H,H-5′a), 3.76 (dd, 1 H, H-5′b), 2.63 (m, 1 H, H-2′a), 2.43 (m, 1 H,H-2′b), 0.91 (s, 18 H, (CH₃)₃CSi), 0.09 (2 s, 12 H, (CH₃)₂Si—);

ToF-MS (ESI): For the molecular ion C₂₉H₄₇N₆O₅Si₂ [M+H]⁺, the calculatedmass was 615.3147, and the observed mass was 615.2288.

N⁶-(2-Nitrobenzyl)-2′-deoxyadenosine (dA.10)

A solution of n-Bu₄NF (216 mg, 0.83 mmol) in THF (1 mL) was added to asolution of compound dA.09 (202 mg, 0.33 mmol) in THF (5 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for two hours. Silica gel 60 (1 g) was added, and the mixturewas evaporated in vacuo to dryness. The residue was purified by silicagel column chromatography to yield N⁶-(2-nitrobenzyl)-2′-deoxyadenosinedA.10 (55 mg, 43%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.48 (br s, 1 H, D2O exchangeable, 6-NH),8.41 (s, 1H, H-8), 8.16 (s, 1 H, H-2), 8.04 (dd, 1 H, Ph-H), 7.66 (d, 1H, Ph-H), 7.51 (m, 2 H, Ph-H), 6.35 (t, 1 H, J=6.4 Hz, H-1′), 5.32 (d, 1H, D₂O exchangeable, 3′-OH), 5.17 (t, 1 H, D₂O exchangeable, 5′-OH),4.97 (bs, 2 H, Ph-CH₂), 4.41 (m, 1 H, H-4′), 3.87 (m, 1 H, H-3′), 3.60(m, 1 H, H-5′a), 3.52 (m, 1 H, H-5′b), 2.71 (m, 1 H, H-2′a), 2.28 (m, 1H, H-2′b);

ToF-MS (ESI): For the molecular ion C₁₇H₁₉N₆O₅ [M+H]⁺, the calculatedmass was 387.1417, and the observed mass was 387.1186.

N⁶-(2-Nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate (WW1p129)

POCl₃ (19 μL, 0.2 mmol) was added to a solution of compound dA.10 (52mg, 0.13 mmol) in trimethylphosphate (0.5 mL) and maintained at minus20-30° C. for 2.5 hours. A solution of bis-tri-n-butylammoniumpyrophosphate (308 mg, 0.65 mmol) and tri-n-butylamine (130 μL) inanhydrous DMF (1.3 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to giveN⁶-(2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate WW1p129 (53 mg,60%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.42 (s, 1 H, H-8), 8.13 (s, 1 H, H-2), 8.09(d, 1 H, Ph-H), 7.55 (m, 2 H, Ph-H), 7.45 (m, 1 H, Ph-H), 6.46 (t, 1 H,J=6.4 Hz, H-1′), 5.05 (bs, 2 H, Ph-CH₂), 4.29 (s, 1 H, H-3′), 4.21 (m, 2H, H-5′a and H-5′b), 2.78 (m, 1 H, H-2′a), 2.59 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.86 (d, J=16.2 Hz), −10.78 (d, J=16.2 Hz),−19.22 (t, J=16.2 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₂N₆O₁₄P₃ [M-H]⁻, the calculatedmass was 625.0250, and the observed mass was 625.0231.

The triphosphate was further purified using preparative HPLC without UVdetection to give sample free from contamination of the naturalnucleotide. Determination of the concentration of the triphosphatesolution was performed by UV/VIS measurement using the extinctioncoefficient of ε₂₆₀=20,800.

Synthesis of N⁶-(4-methoxy-2-nitrobenzyl)-2′-deoxyadenosine triphosphate(WW2p005)

Scheme. Synthesis ofN⁶-(4-methoxy-2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate. (i) NaH,DMF, 4-methoxy-2-nitrobenzyl bromide, 0° C., then gradually warmed toroom temperature, 64%; (ii) SiO₂, high vacuum, 70-80° C., 24 hours, 84%;(iii) n-Bu₄NF, THF, 99%; (iv) POCl₃, (MeO)₃PO, minus 20-30° C.;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃; 40%.

N⁶-tert-Butyloxycarbonyl-N⁶-(4-methoxy-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.11)

NaH (26 mg, 1.1 mmol, dry) was added to a solution of compound dA.07(580 mg, 1.0 mmol) in anhydrous DMF (5 mL) at 0° C. and stirred for 45minutes. A solution of 4-methoxy-2-nitrobenzyl bromide (260 mg, 1.05mmol) in anhydrous DMF (1.0 mL) was added dropwise. The mixture wasgradually warmed to room temperature and stirred overnight. DMF wasremoved in vacuo, and the residue was dissolved in ethyl acetate (50 mL)and washed twice with saturated NH₄Cl solution (30 mL each). Thecombined aqueous layer was extracted with ethyl acetate (20 mL), and thecombined organic layer was dried over Na₂SO₄, concentrated in vacuo, andpurified by silica gel column chromatography to yieldN6-tert-butyloxycarbonyl-N⁶-(4-methoxy-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.11 (480 mg, 64%) as a viscous oil.

¹H NMR (400 MHz, CDCl₃): δ 8.68 (s, 1 H, H-8), 8.37 (s, 1 H, H-2), 7.66(d, 1 H, J=8.7 Hz, Ph-H), 7.55 (d, 1 H, J=2.7 Hz, Ph-H), 7.10 (dd, 1 H,J=2.7 and 8.7 Hz, Ph-H), 6.51 (t, 1 H, J=6.4 Hz, H-1′), 5.55 (s, 2 H,Ph-CH₂), 4.63 (m, 1 H, H-4′), 4.03 (m, 1H, H-3′), 3.87 (m, 1 H, H-5′a),3.84 (s, 3 H, OCH₃), 3.78 (m, 1 H, H-5′b), 2.63 (m, 1 H, H-2′a), 2.44(m, 1 H, H-2′b), 1.41 (s, 9 H, (CH₃)₃CO), 0.92 (s, 18 H, (CH₃)₃CSi),0.10 (2 s, 12 H, (CH₃)₂Si—);

ToF-MS (ESI): For the molecular ion C₃₅H₅₇N₆O₈Si₂ [M+H]⁺, the calculatedmass was 745.3776, and the observed mass was 745.3782.

N⁶-(4-Methoxy-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.12)

Silica gel 60 (5 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for 24 hours) was added to a solution of compounddA.11 (530 mg, 0.71 mmol) in CH₂Cl₂ (30 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 70-80° C.under reduced pressure for two days, washed three times with methanol(20 mL each), and filtered using a buchi funnel. The combined filtratewas concentrated in vacuo and purified by silica gel columnchromatography to yieldN⁶-(4-methoxy-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.12 (385 mg, 84%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.37 (s, 1 H, H-8), 8.08 (s, 1 H, H-2), 7.66(d, 1 H, J=8.5 Hz, Ph-H), 7.57 (m, 1 H, J=2.7 Hz Ph-H), 7.09 (dd, 1 H,J=2.7 and 8.7 Hz Ph-H), 6.52 (t, 1 H, 6-NH), 6.43 (t, 1 H, J=6.4 Hz,H-1′), 5.07 (bs, 2 H, Ph-CH₂), 4.60 (m, 1 H, H-4′), 4.00 (m, 1 H, H-3′),3.87 (m, 1 H, H-5′a), 3.85 (s, 3 H, OCH₃), 3.76 (dd, 1 H, H-5′b), 2.62(m, 1 H, H-2′a), 2.43 (m, 1 H, H-2′b), 0.91 (s, 18 H, (CH₃)₃CSi), 0.09(2 s, 12 H, (CH₃)₂Si—);

ToF-MS (ESI): For the molecular ion C₃₀H₄₈N₆O₆Si₂ [M+H]⁺, the calculatedmass was 645.3252, and the observed mass was 645.3248.

N⁶-(4-Methoxy-2-nitrobenzyl)-2′-deoxyadenosine (dA.13)

A solution of n-Bu₄NF (353 mg, 1.35 mmol) in THF (2 mL) was added to asolution of compound dA.12 (350 mg, 0.54 mmol) in THF (5 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for four hours. Silica gel 60 (1.5 g) was added, and the mixturewas evaporated in vacuo to dryness. The residue was purified by silicagel column chromatography to yieldN⁶-(4-methoxy-2-nitrobenzyl)-2′-deoxyadenosine dA.13 (225 mg, 99%) as ayellow foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.40 (bs, 1 H, D₂O exchangeable, 6-NH),8.39 (s, 1H, H-8), 8.15 (s, 1 H, H-2), 7.54 (d, 1 H, J=2.7 Hz, Ph-H),7.44 (d, 1 H, J=8.2 Hz, Ph-H), 7.24 (d, 1 H, J=2.7 and 8.7 Hz, Ph-H),6.33 (t, 1 H, J=6.7 Hz, H-1′), 5.32 (d, 1H, D₂O exchangeable, 3′-OH),5.17 (t, 1 H, D₂O exchangeable, 5′-OH), 4.88 (bs, 2 H, Ph-CH₂), 4.40 (m,1 H, H-4′), 3.87 (m, 1 H, H-3′), 3.81 (s, 3 H, OCH₃), 3.60 (m, 1 H,H-5′a), 3.52 (m, 1 H, H-5′b), 2.70 (m, 1 H, H-2′a), 2.26 (m, 1 H,H-2′b);

ToF-MS (ESI): For the molecular ion C₁₈H₂₁N₆O₆ [M+H]⁺, the calculatedmass was 417.1523, and the observed mass was 417.1458.

N⁶-(4-Methoxy-2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate (WW2p005)

POCl₃ (19 μL, 0.2 mmol) was added to a solution of compound dA.13 (42mg, 0.1 mmol) in trimethylphosphate (0.5 mL) and maintained at minus20-30° C. for three hours. A solution of bis-tri-n-butylammoniumpyrophosphate (237 mg, 0.5 mmol) and tri-n-butylamine (100 μL) inanhydrous DMF (1 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred at room temperature for one hour and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to giveN⁶-(4-methoxy-2-nitrobenzyl)-2′-deoxyadenosine-5′-triphosphate WW2p005(28 mg, 40%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.41 (s, 1 H, H-8), 8.17 (s, 1 H, H-2), 7.64(d, 1 H, J=2.7 Hz, Ph-H), 7.47 (d, 1 H, J=8.7 Hz, Ph-H), 7.15 (d, 1 H,J=2.7 and 8.7 Hz, Ph-H), 6.46 (t, 1 H, J=6.7 Hz, H-1′), 4.97 (bs, 2 H,Ph-CH₂), 4.29 (s, 1 H, H-3′), 4.20 (m, 2 H, H-5′a and H-5′b), 3.84 (s, 3H, OCH₃), 2.80 (m, 1 H, H-2′a), 2.60 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.97 (d, J=19.9 Hz), −11.07 (d, J=19.3 Hz),−21.76 (t, J=19.3 Hz);

ToF-MS (ESI): For the molecular ion C₁₈H₂₁N₆O₁₅P₃Na [M-2H+Na]⁻, thecalculated mass was 677.0175, and the observed mass was 677.0197.

The triphosphate was further purified using preparative HPLC without UVdetection to give sample free from contamination of the naturalnucleotide. Determination of the concentration of the triphosphatesolution was performed by UV/VIS measurement using the extinctioncoefficient of ε₂₆₀=20,800.

Synthesis of 6-FAM LabeledN⁶-[4-(3-amino-1-propyl)-2-nitrobenzyl]-2′-deoxyadenosine triphosphate(WW2p055)

Scheme. Synthesis of 6-FAM labeledN⁶-[4-(3-amino-1-propyl)-2-nitrobenzyl]-2′deoxyadenosine triphosphate.(i) NaH, DMF, 4-iodo-2-nitrobenzyl bromide, 0° C., then gradually warmedto room temperature, 61%; (ii) PdCl₂(PPh₃)₂, CuI, Et₃N, THF, reflux,94%; (iii) SiO₂, vacuum, 70-80° C., 82%; (iv) n-Bu₄NF, THF, 0° C., thengradually warmed to room temperature, 33%; (v) POCl₃, proton sponge,(MeO)₃PO, minus 20-30° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, twominutes; 1 M HNEt₃HCO₃, one hour; NH₄OH, one hour; 72%; (vi) 6-FAM-SE,0.1 M NaHCO₃/NaCO₃, pH 9.2.

N⁶-tert-Butyloxycarbonyl-N⁶-(4-iodo-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.14)

NaH (40 mg, 1.66 mmol, dry) was added to a solution of compound dA.07(875 mg, 1.51 mmol) in anhydrous DMF (10 mL) at 0° C. and stirred for 45minutes. A solution of 4-iodo-2-nitrobenzyl bromide (516 mg, 1.51 mmol)in anhydrous DMF (2 mL) was added dropwise. The mixture was graduallywarmed to room temperature and stirred for four hours. DMF was removedin vacuo, and the residue was dissolved in ethyl acetate (50 mL), washedwith saturated NH₄Cl solution (30 mL), and washed twice with water (30mL each). The combined aqueous layer was extracted with ethyl acetate(20 mL), and the combined organic layer was dried over Na₂SO₄,concentrated in vacuo, and purified by silica gel column chromatographyto yieldN⁶-tert-butyloxycarbonyl-N⁶-(4-iodo-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.14 (777 mg, 61%) as a white foam.

¹H NMR (400 MHz, CDCl₃) δ 8.66 (s, 1 H, H-8), 8.38 (s, 1 H, H-2), 8.34(d, 1 H, J=1.1 Hz, Ph-H), 7.85 (dd, 1 H, J=1.1 and 8.3 Hz, Ph-H), 7.50(d, 1 H, J=8.3 Hz, Ph-H), 6.50 (t, 1 H, J=6.3 Hz, H-1′), 5.54 (s, 2 H,Ph-CH₂), 4.63 (m, 1 H, H-3′), 4.03 (m, 1 H, H-4′), 3.87 (m, 1 H, H-5′a),3.78 (m, 1 H, H-5′b), 2.62 (m, 1 H, H-2′a), 2.46 (m, 1 H, H-2′b), 1.41(s, 9 H, (CH₃)₃CO), 0.92 (s, 18 H, (CH₃)₃CSi), 0.10 (2 s, 12 H,(CH₃)₂Si).

N⁶-tert-Butyloxycarbonyl-N⁶-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.15)

Under a N₂ atmosphere, a mixture of compound dA.14 (730 mg, 0.87 mmol),N-propargyltrifluoroacetamide (183 mg, 1.2 mmol), CuI (33 mg, 0.17mmol), bis(triphenylphosphine)palladium(II) chloride (61 mg, 0.087mmol), and Et₃N (1.6 mL, 11.57 mmol) in anhydrous THF (7.5 mL) wasrefluxed for six hours in the dark. The mixture was concentrated invacuo, and the residue was purified by silica gel column chromatographyto yieldN⁶-tert-butyloxycarbonyl-N⁶-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosinedA.15 (706 mg, 94%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.67 (s, 1 H, H-8), 8.39 (s, 1 H, H-2), 8.06(d, 1 H, J=1.5 Hz, Ph-H), 7.73 (d, 1 H, J=8.2 Hz, Ph-H), 7.56 (dd, 1 H,J=1.5 and 8.2 Hz, Ph-H), 7.28 (br s, 1 H, NH), 6.51 (t, 1 H, J=6.4 Hz,H-1′), 5.59 (s, 2 H, Ph-CH₂), 4.63 (m, 1 H, H-3′), 4.37 (m, 2 H, CH₂),4.03 (m, 1 H, H-4′), 3.87 (m, 1 H, H-5′a), 3.78 (m, 1 H, H-5′b), 2.63(m, 1 H, H-2′a), 2.48 (m, 1 H, H-2′b), 1.40 (s, 9 H, (CH₃)₃CO), 0.92 (s,18H, (CH₃)₃CSi), 0.10 (2 s, 12 H, (CH₃)₂Si—).

N⁶-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyadenosine(dA.16)

Silica gel 60 (3.5 g, 100-200 mesh, activated by heating to 70-80° C.under reduced pressure for 24 hours) was added to a solution of compounddA.15 (507 mg, 0.59 mmol) in CH₂Cl₂ (30 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 70-80° C.under reduced pressure for 42 hours, washed three times with methanol(20 mL each), and filtered using a buchi funnel. The combined filtratewas concentrated in vacuo and purified by silica gel columnchromatography to yieldN⁶-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethyl-silyl-2′-deoxyadenosinedA.16 (366 mg, 82%) as a yellow foam.

¹H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1 H, H-8), 8.12 (s, 1 H, H-2), 8.05(d, 1 H, J=1.3 Hz, Ph-H), 7.66 (d, 1 H, J=8.0 Hz, Ph-H), 7.48 (dd, 1 H,J=1.3 and 8.0 Hz, Ph-H), 7.20 (bs, 1 H, NH), 6.54 (t, 1 H, NH), 6.44 (t,1 H, J=6.4 Hz, H-1′), 5.16 (bs, 2 H, Ph-CH₂), 4.61 (m, 1 H, H-4′), 4.39(d, 2 H, CH₂), 4.00 (m, 1 H, H-3′), 3.87 (m, 1 H, H-5′a), 3.78 (m, 1 H,H-5′b), 2.62 (m, 1 H, H-2′a), 2.44 (m, 1 H, H-2′b), 1.40 (s, 9 H,(CH₃)₃CO), 0.91 (s, 18 H, (CH₃)₃CSi), 0.09 (2 s, 12 H, (CH₃)₂Si—).

N⁶-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxyadenosine(dA.17)

A solution of n-Bu₄NF (282 mg, 1.08 mmol) in THF (2 mL) was added to asolution of compound dA.16 (330 mg, 0.43 mmol) in THF (5 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for two hours. Methanol (5 mL) and silica gel 60 (2 g) wereadded, and the mixture was evaporated in vacuo to dryness. The residuewas purified by silica gel column chromatography to yieldN⁶-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxyadenosinedA.17 (75 mg, 33%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 10.08 (t, 1 H, D₂O exchangeable, NH), 8.50(br s, 1H, D₂O exchangeable, NH), 8.42 (s, 1 H, H-8), 8.16 (s, 1 H,H-2), 8.06 (d, 1 H, J=1.6 Hz, Ph-H), 7.71 (dd, 1 H, J=1.6 and 8.1 Hz,Ph-H), 7.51 (m, 1 H, Ph-H), 6.35 (t, 1 H, J=6.4 Hz, H-1′), 5.30 (d, 1 H,D₂O exchangeable, 3′-OH), 5.13 (br s, 1 H, D₂O exchangeable, 5′-OH),4.96 (br s, 2 H, Ph-CH₂), 4.41 (m, 1 H, H-4′), 4.29 (d, 2 H, CH₂), 3.87(m, 1 H, H-3′), 3.60 (m, 1 H, H-5′a), 3.51 (m, 1 H, H-5′b), 2.72 (m, 1H, H-2′a), 2.27 (m, 1 H, H-2′b).

N⁶-[4-(3-Amino-1-propyl)-2-nitrobenzyl]-2′-deoxyadenosine-5′-triphosphate(dA.18)

POCl₃ (8.5 μL, 0.09 mmol) was added to a solution of compound dA.17 (32mg, 0.06 mmol) and proton sponge (19 mg, 0.09 mmol) intrimethylphosphate (0.5 mL) and maintained at minus 20-30° C. for twohours. A solution of bis-tri-n-butylammonium pyrophosphate (142 mg, 0.3mmol) and tri-n-butylamine (60 μL) in anhydrous DMF (0.6 mL) was added.After two minutes of stirring, triethylammonium bicarbonate buffer (1 M,pH 7.5; 5 mL) was added. The reaction was stirred for one hour at roomtemperature, followed by the dropwise addition of concentrated ammoniumhydroxide (10 mL, 27%) at 0° C. The mixture was stirred for anadditional hour at room temperature and then lyophilized to dryness. Theresidue obtained was dissolved in water (10 mL), filtered, and purifiedby anion exchange chromatography using a Q Sepharose FF column (2.5×20cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes)at a flow rate of 4.5 mL/min. The fractions containing triphosphate werecombined and lyophilized to give triphosphate dA.18 (31 mg, 72%) as awhite fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.47 (s, 1 H, H-8), 8.23 (s, 1 H, Ph-H), 8.20(s, 1 H, H-2), 7.65 (d, 1 H, J=8.2 Hz, Ph-H), 7.57 (d, 1 H, J=8.2 Hz,Ph-H), 6.52 (t, 1 H, J=6.8 Hz, H-1′), 5.14 (br s, 2 H, Ph-CH₂), 4.31 (s,1 H, H-4′), 4.21 (m, 2 H, H-5′a and H-5′b), 3.60 (s, 2 H, CH₂), 2.82 (m,1 H, H-2′a), 2.62 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.43 (d, J=15.4 Hz), −10.46 (d, J=15.6 Hz),−18.85 (t, J=15.6 Hz);

ToF-MS (ESI): For the molecular ion C₂₀H₂₃N₇O₁₄P₃ [M-H]⁻, the calculatedmass was 678.0516, and the observed mass was 678.0857.

6-FAM LabeledN⁶-[4-(3-Amino-1-propyl)-2-nitrobenzyl]-2′-deoxyadenosine-5′-triphosphate(WW2p055)

A solution of 6-FAM-SE (6.7 mg, 0.014 mmol) in anhydrous DMSO (70 μL)was added to a solution of triphosphate dA.18 (4.4 μmol) inNa₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2; 3 mL) and incubated at roomtemperature for one hour. The reaction was purified by reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield the6-FAM labeled triphosphate WW2p055 (2.6 mg, 49%). Mobile phase: A, 100mM triethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). Elution was performed with a linear gradient of5-20% B for 20 minutes and then 20-90% B for 20 minutes. Theconcentration of WW2p055 was estimated by adsorption spectroscopy usingthe extinction coefficient of the 6-FAM dye (i.e., 68,000 at 494 nm).

³¹P NMR (162 MHz, D₂O): δ-5.87 (d, J=19.8 Hz), −11.01 (d, J=19.1 Hz),−21.76 (t, J=19.8 Hz);

ToF-MS (ESI): For the molecular ion C₄₁H₃₅N₇O₂₀P₃ [M+H]⁺, the calculatedmass was 1038.1150, and the observed mass was 1138.1281.

Synthesis of 5(6)-SFX labeledN⁶-[4-(3-amino-1-propyl)-2-nitrobenzyl]-2′deoxy-adenosine triphosphate(WW2p052)

Scheme. Synthesis of 5(6)-SFX labeledN⁶-[4-(3-amino-1-propyl)-2-nitrobenzyl]-2′-deoxyadenosine triphosphate.(i) 5(6)-SFX, 0.1 M NaHCO₃/NaCO₃, pH 9.2.

5(6)-SFX labeledN⁶-[4-(3-Amino-1-propyl)-2-nitrobenzyl]-2′-deoxyadenosine-5′-triphosphate(WW2p052)

A solution of 5(6)-SFX (1.5 mg, 2.55 μmol) in anhydrous DMSO (30 μL) wasadded to a solution of triphosphate dA.18 (0.54 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 0.8 mL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 5(6)-SFX labeledtriphosphate WW2p052. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). Elutionwas performed with a linear gradient of 5-20% B for 20 minutes and then20-90% B for 20 minutes. The concentration of WW2p052 was estimated byadsorption spectroscopy using the extinction coefficient of the 6-FAMdye (i.e., 68,000 at 494 nm).

Synthesis of N⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyadenosine triphosphate(WW3p006)

Scheme. Synthesis of N⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyadenosinetriphosphate (i) 1-(2-nitrophenyl)ethylamine, BOP, DIPEA, DMF, roomtemperature, overnight, 42%; (ii) POCl₃, proton sponge, (MeO)₃PO, 0° C.,two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃,one hour.

N⁶-[1-(2-Nitrophenyl)ethyl]-2′-deoxyadenosine (dA.19)

To a suspension of 2′-deoxyinosine (100 mg, 0.4 mmol) andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate(BOP, 210 mg, 0.48 mmol) in anhydrous DMF (1 mL),N,N-diisopropylethylamine (100 μL, 0.6 mmol) was added followed by theaddition of a solution of 1-(2-nitrophenyl)ethylamine (250 mg, 1.51mmol) in DMF (1 mL). The reaction was stirred at room temperature for 64hours. Silica gel 60 (1 g, 60-200 mesh) was added, and the mixture wasevaporated in vacuo to dryness. The residue was purified by silica gelcolumn chromatography to yieldN⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyadenosine dA.19 (67 mg, 42%, 1:1mixture of diastereomers) as a white foam.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.68 (br s, 1 H, D₂Oexchangeable, NH), 8.42 (br s, 1 H, H-8), 8.16 and 8.06 (2 s, 1 H, H-2),7.88 (m, 2 H, Ph-H), 7.69 (m, 1 H, Ph-H), 7.46 (m, 1 H, Ph-H), 6.34 (m,1 H, H-1′), 5.79 (m, 1 H, Ph-CH), 5.32 (br s, 1 H, D₂O exchangeable,3′-OH), 5.16 (br s, 1 H, D₂O exchangeable, 5′-OH), 4.42 (m, 1 H, H-3′),3.88 (m, 1 H, H-4′), 3.61 (m, 1 H, H-5′a), 3.53 (m, 1 H, H-5′b), 2.71(m, 2 H, H-2′a), 2.25 (m, 1 H, H-2′b), 1.68 (d, 3 H, J=6.8 Hz, CH₃)

N⁶-[1-(2-Nitrophenyl)ethyl]-2′-deoxyadenosine-5′-triphosphate (WW3p006)

Compound dA.19 (30 mg, 0.075 mmol) and proton sponge (32 mg, 0.15 mmol)were evaporated three times from anhydrous pyridine (2 mL) and dissolvedin trimethylphosphate (0.5 mL). POCl₃ (10.5 μL, 0.11 mmol) was added,and the mixture was stirred for two hours at 0° C. A solution ofbis-tri-n-butylammonium pyrophosphate (178 mg, 0.38 mmol) andtri-n-butylamine (75 μL) in anhydrous DMF (0.75 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and part of the solution was purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yield N⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyadenosine-5′-triphosphateWW3p006 (1:1 mixture of diastereomers). Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). Elution was performed with a linear gradient of5-50% B for 40 minutes and then 50-90% B for 10 minutes.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 8.47 (s, 1 H, H-8), 8.12 (2s, 1 H, H-2), 8.02 (d, 1 H, J=8.2 Hz, Ph-H), 7.78 (d, 1 H, J=7.8 Hz,Ph-H), 7.67 (t, 1 H, J=7.6 Hz, Ph-H), 7.49 (t, 1 H, J=8.1 Hz, Ph-H),6.49 (t, 1 H, J=6.4 Hz, H-1′), 5.89 (bs, 1H, Ph-CH), 4.29 (m, 1 H,H-4′), 4.23-4.15 (m, 2 H, H-5′a and H-5′b), 2.81 (m, 1 H, H-2′a), 2.58(m, 1 H, H-2′b), 1.74 (d, 3 H, J=6.8 Hz, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: δ-5.65 (m), −10.52 (d, J=19.6Hz), −21.32 (m);

ToF-MS (ESI): For the molecular ion C₁₈H₂₁N₆O₁₄P₃Na [M-2H+Na]⁻, thecalculated mass was 661.0226, and the observed mass was 661.0492.

Synthesis of 6-FAM labeledN⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosinetriphosphate (WW3p015)

Scheme. Synthesis of 6-FAM labeledN⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosinetriphosphate. (i) 1-(4-iodo-2-nitrophenyl)ethylamine, BOP, DIPEA, DMF,room temperature, 65%; (ii) N-propargyltrifluoroacetamide, Pd(PPh₃)₄(0),CuI, Et₃N, anhydrous DMF, 4.5 h, 94%; (iii) POCl₃, proton sponge,(MeO)₃PO, 0° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes;1 M HNEt₃HCO₃, one hour; NH₄OH, one hour; (iv) 6-FAM-SE, 0.1 MNaHCO₃/Na₂CO₃, pH 9.2, one hour.

N⁶-[1-(4-Iodo-2-nitrophenyl)ethyl]-2′-deoxyadenosine (dA.20)

To a suspension of 2′-deoxyinosine (150 mg, 0.6 mmol) andbenzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphonate(BOP, 379 mg, 0.86 mmol) in anhydrous DMF (1.5 mL),N,N-diisopropylethylamine (186 μL, 1.1 mmol) was added followed by theaddition of a solution of 1-(4-iodo-2-nitrophenyl)ethylamine (460 mg,1.57 mmol) in anhydrous DMF (0.5 mL). The mixture was stirred undernitrogen atmosphere for 48 hours. DMF was removed in vacuo, and thecrude product was purified by silica gel column chromatography to yieldN⁶-[1-(4-iodo-2-nitrophenyl)ethyl]-2′-deoxyadenosine dA.20 (204 mg, 65%,1:1 mixture of diastereomers) as a white foam.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 8.72 (br m, 1 H, D₂Oexchangeable, NH), 8.42 (br s, 1 H, H-8), 8.20 (s, 1 H, H-2), 8.06 (m, 2H, Ph-H), 7.65 (m, 1 H, Ph-H), 6.34 (m, 1 H, H-1′), 5.70 (br s, 1 H,PhCH), 5.32 (br s, 1 H, D₂O exchangeable, 3′-OH), 5.15 (br m, 1 H, D₂Oexchangeable, 5′-OH), 4.42 (m, 1 H, H-4′), 3.88 (m, 1 H, H-3′), 3.61 (m,1 H, H-5′a), 3.53 (m, 1 H, H-5′b), 2.71 (m, 1 H, H-2′a), 2.25 (m, 1 H,H-2′b), 1.65 (d, 3 H, J=6.9 Hz, CH₃);

¹³C NMR (100 MHz, MeOH-d₄) for diastereomers: δ 153.39 (CH), 151.94 (C),150.65 (C), 143.44 (CH), 141.82 (C), 141.40 (C), 133.77/133.69 (CH),130.55/130.52 (CH), 121.37 (C), 92.09 (C), 91.87 (C), 89.97 (CH), 87.21(CH), 73.18/73.15 (CH), 65.78/65.76 (CH₂), 47.12 (CH), 41.62 (CH₂),37.14/37.10 (CH₃);

ES-MS (ESI): m/e 525 [M-H]⁻.

N⁶-{1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine(dA.21)

A solution of compound dA.20 (108 mg, 0.21 mmol),N-propargyltrifluoroacetamide (127 mg, 0.84 mmol), CuI (11 mg, 0.06mmol), tetrakis(triphenylphosphine)-palladium(0) (33 mg, 0.03 mmol), andEt₃N (80 μL, 0.56 mmol) in anhydrous DMF (1.5 mL) was stirred at roomtemperature for four and a half hours. The mixture was concentrated invacuo and purified by silica gel column chromatography to yieldN⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosinedA.21 (106 mg, 94%, 1:1 mixture of diastereomers) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.10 (br m, 1 H, D₂Oexchangeable, NH), 8.71 (br m, 1 H, D₂O exchangeable, NH), 8.43 (br s, 1H, H-8), 8.15 and 8.06 (2 s, 1 H, H-2), 7.93 (s, 1 H, Ph-H), 7.86 (m, 1H, Ph-H), 7.75 (m, 1 H, Ph-H), 6.35 (br m, 1 H, H-1′), 5.73 (br m, 1 H,Ph-CH), 5.31 (br s, 1 H, D₂O exchangeable, 3′-OH), 5.15 (br m, 1 H, D₂Oexchangeable, 5′-OH), 4.40 (m, 1 H, H-4′), 4.31 (d, 2 H, J=5.3 Hz, CH₂),3.88 (m, 1 H, H-3′), 3.62 (m, 1 H, H-5′a), 3.51 (m, 1 H, H-5′b), 2.71(m, 1 H, H-2′a), 2.25 (m, 1 H, H-2′b), 1.67 (d, 3 H, J=6.8 Hz, CH₃);

¹³C NMR (100 MHz, MeOH-d₄) for diastereomers: δ 157.85/157.48 (C),152.26 (CH), 149.50 (C), 140.27 (C), 136.04 (CH), 128.02 (CH), 127.07(CH), 122.56 (C), 120.25 (C), 117.81 (C), 114.96 (C), 88.85 (CH), 86.08(CH), 85.81 (C), 80.67 (C), 72.07/72.04 (CH), 62.66/62.63 (CH₂), 48.30(CH), 40.47 (CH₂), 29.46 (CH₂), 24.26 (CH₃);

N⁶-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(dA.22)

Compound dA.21 (44 mg, 0.08 mmol) and proton sponge (34 mg, 0.16 mmol)were evaporated three times from anhydrous pyridine (2 mL) and dissolvedin trimethylphosphate (0.5 mL). POCl₃ (11 μL, 0.12 mmol) was added, andthe mixture was stirred for two hours at 0° C. A solution ofbis-tri-n-butylammonium pyrophosphate (190 mg, 0.4 mmol) andtri-n-butylamine (80 μL) in anhydrous DMF (0.8 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and part of the solution was purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yieldN⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved using a linear gradient of 5-50% B for 20 minutes and then50-90% B for 10 minutes.N⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphatewas then treated with concentrated ammonium hydroxide (1 mL, 27%) atroom temperature for one hour to yieldN⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphatedA.22 (1:1 mixture of diastereomers).

¹H NMR (400 MHz, D₂O): δ 8.45 (s, 1 H, H-8), 8.08 (2 s, 1 H, H-2), 7.95(s, 1 H, Ph-H), 7.65 (m, 1 H, Ph-H), 7.53 (m, 1 H, Ph-H), 6.45 (t, 1 H,J=6.4 Hz, H-1′), 5.80 (br s, 1 H, Ph-CH), 4.28 (s, 1 H, H-4′), 4.18 (m,2 H, H-5′a and H-5′b), 3.64 (s, 2 H, CH₂), 2.78 (m, 1 H, H-2′a), 2.57(m, 1 H, H-2′b), 1.69 (d, 3 H, J=6.8 Hz, CH₃);

³¹P NMR (162 MHz, D₂O): δ-5.29 (d, J=20.1 Hz), −10.45 (d, J=19.1 Hz),−21.08 (t, J=19.6 Hz);

ToF-MS (ESI): For the molecular ion C₂₁H₂₅N₇O₁₄P₃ [M-H]⁻, the calculatedmass was 692.0672 and the observed mass was 692.0757.

6-FAM LabeledN⁶-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(WW3p015)

A solution of 6-FAM-SE (1.5 mg, 3.15 μmol) in anhydrous DMSO (30 μL) wasadded to a solution of triphosphate dA.22 (0.34 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 100 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-FAM labeledtriphosphate WW3p015. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLCpurification was achieved using a linear gradient of 5-50% B for 40minutes and then 50-90% B for 10 minutes. The concentration of WW3p015was estimated by adsorption spectroscopy using the extinctioncoefficient of the 6-FAM dye (i.e., 68,000 at 494 nm).

Separation of DiastereoisomersN⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-ethyl}-2′-deoxyadenosine-5′-triphosphate(dA.22 dS1 and dA.22 ds2)

Separation of the two diastereoisomers of dA.22 was performed byreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yield N⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosinetriphosphate dA.22 dS1 (single diastereoisomer, absolute configurationnot determined) and N⁶-{(S orR)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosinetriphosphate dA.22 dS2 (single diastereoisomer, absolute configurationnot determined). Mobile phase: A, 100 mM triethylammonium acetate (TEAA)in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLCpurification was achieved using a linear gradient of 5-25% B for 50minutes and then 25-50% B for 30 minutes.

Synthesis of 6-FAM Labeled Single Diastereoisomer N⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(WW3p021)

Scheme. Synthesis of 6-FAM labeled single diastereoisomer N⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate.(i) 6-FAM-SE, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour.

6-FAM Labeled Single Diastereoisomer N⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyadenosine-5′-triphosphate(WW3p021)

A solution of 6-FAM-SE (0.75 mg, 1.57 μmol) in anhydrous DMSO (15 μL)was added to a solution of triphosphate dA.22 ds1 (0.26 μmol, singlediastereoisomer, absolute configuration not determined) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 150 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-FAM labeled singlediastereoisomer triphosphate WW3p021. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved using a lineargradient of 5-50% B for 40 minutes and then 50-90% B for 10 minutes. Theconcentration of WW3p021 was estimated by absorption spectroscopy usingthe extinction coefficient of the 6-FAM dye (i.e., 68,000 at 494 nm).

Synthesis of 6-FAM Labeled Single Diastereoisomer N⁶-{(R orS)-1-{4-[3-(6-aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyadenosine-5′-triphosphate(WW3p032)

Scheme. Synthesis of 6-FAM labeled single diastereoisomer N⁶-{(R orS)-1-{4-[3-(6-aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyadenosinetriphosphate. (i) 6-N-(trifluoroacetyl)aminocaproic acid N-succinimidylester, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour; NH4OH, one hour; (ii)6-FAM-SE, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour.

N⁶-{(R orS)-1-{4-[3-(6-Aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyadenosine-5′-triphosphate(Single Diastereoisomer dA.23 ds1)

A solution of 6-N-(trifluoroacetyl)aminocaproic acid N-succinimidylester (0.5 mg, 1.54 μmol) in anhydrous DMSO (10 μL) was added to asolution of triphosphate dA.22 ds1 (0.25 μmol, single diastereoisomer,absolute configuration not determined) in Na₂CO₃/NaHCO₃ buffer (0.1 M,pH 9.2; 200 μL) and incubated at room temperature for one hour. NH₄OH(500 uL, 25% aq) was added and the mixture was incubated at roomtemperature for another hour. The reaction was purified by reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yieldtriphosphate dA.23 ds1 (single diastereoisomer, absolute configurationnot determined). Mobile phase: A, 100 mM triethylammonium acetate (TEAA)in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLCpurification was achieved using a linear gradient of 5-50% B for 20minutes and then 50-90% B for 10 minutes.

Synthesis of 6-FAM Labeled Single Diastereoisomer N⁶-{(R orS)-1-{4-[3-(6-aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyadenosine-5′-triphosphate(WW3p032)

A solution of 6-FAM-SE (0.5 mg, 1.05 μmol) in anhydrous DMSO (10 μL) wasadded to a solution of triphosphate dA.23 ds1 (0.196 μmol, singlediastereoisomer, absolute configuration not determined) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 200 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-FAM labeled singlediastereoisomer triphosphate WW3p032. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved using a lineargradient of 5-50% B for 40 minutes and then 50-90% B for 10 minutes. Theconcentration of WW3p032 was estimated by absorption spectroscopy usingthe extinction coefficient of the 6-FAM dye (i.e., 68,000 at 494 nm).

Example 2 dT Compounds Synthesis OfN³-(2-nitrobenzyl)-thymidine-5′-triphosphate (WW1P050)

Scheme. Synthesis of N³-(2-nitrobenzyl)-thymidine-5′-triphosphate. (i)TBSCl, imidazole, anhydrous CH₂Cl₂, room temperature, overnight, 58%;(ii) 2-nitrobenzyl bromide, n-Bu₄NOH, NaI, NaOH (1 M), CHCl₃, roomtemperature, overnight, 37%; (iii) n-Bu₄NF, THF, 25° C., 45 minutes,80%; (iv) POCl₃, proton sponge, (MeO)₃PO, 0° C., six hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, one hour,56%.

5′-O-tert-butyldimethylsilyl-thymidine (dT.01)

A solution of thymidine dT (2.85 g, 11.76 mmol), imidiazole (2.44 g,35.80 mmol) and TBSCl (1.77 g, 11.76 mmol) in anhydrous CH₂Cl₂ (20 mL)was stirred at room temperature overnight under a N₂ atmosphere. Thereaction mixture was then concentrated in vacuo to a viscous oil,followed by the addition of ethyl ether (60 mL) and water (60 mL). Theorganic layer was separated and washed twice with water (20 mL each),and the combined aqueous layer was extracted with ethyl ether (20 mL).The combined organic layer was dried over Na₂SO₄, concentrated in vacuoand purified by silica gel column chromatography to give5′-O-tert-butyldimethylsilyl-thymidine dT.01 (3.44 g, 82%) as a whitesolid.

¹H NMR (400 MHz, CDCl₃): δ 9.35 (br s, 1 H, H-3), 7.53 (d, 1 H, J=1.2Hz, H-6), 6.39 (dd, 1 H, J=8.3, 5.6 Hz, H-1′), 4.45 (m, 1 H, H-4′), 4.07(m, 1 H, H-3′), 3.87 (m, 2 H, H-5′a and H-5′b), 2.99 (br. s, 1 H,3′-OH), 2.38 (m, 1 H, H-2′b), 2.09 (m, 1 H, H-2′b), 1.91 (d, 3 H, J=1.2Hz, 5-Me), 0.92 (s, 9 H, (CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si).

N³-(2-Nitrobenzyl)-5′-O-tert-butyldimethylsilyl-thymidine (dT.02)

To a vigorously stirred mixture of compound dT.01 (660 mg, 1.85 mmol),tetrabutylammonium hydroxide (0.5 mL), sodium iodide (55 mg) in CHCl₃ (5mL), and NaOH (1 M; 5 mL), a solution of 2-nitrobenzyl bromide (400 mg,1.85 mmol) in CHCl₃ (5 mL) was added dropwise and stirred at roomtemperature overnight. The organic layer was separated, and the aqueouslayer was extracted twice with chloroform (5 mL each). The combinedorganic layer was washed with water (5 mL), brine (5 mL), and dried overNa₂SO₄. The solvent was evaporated in vacuo, and the residue waspurified by silica gel column chromatography to yieldN³-(2-nitrobenzyl)-5′-O-tert-butyldimethylsilyl-thymidine dT.02 (562 mg,58%) as a white foam.

¹H NMR (CDCl₃): δ 7.98 (dd, 1 H, J=7.2, 1.2 Hz, Ph-H), 7.60 (d, 1 H,J=1.2 Hz, H-6), 7.49 (dt, 1 H, J=7.6, 1.2 Hz, Ph-H), 7.36 (dt, 1 H,J=8.1, 1.4 Hz, Ph-H), 7.16 (dd, 1 H, J=7.8, 1.1 Hz, Ph-H), 6.31 (dd, 1H, J=8.2, 5.7 Hz, H-1′), 5.50 (d, 1 H, J=16.2 Hz, PhCH₂), 5.44 (d, 1 H,J=16.2 Hz, PhCH₂), 4.40 (m, 1 H, H-4′), 3.97 (q, 1 H, J=2.4 Hz, H-3′),3.82 (dq, 2 H, J=11.4, 2.4 Hz, H-5′a and H-5′b), 2.98 (s, 1 H, 3′-OH),2.29 (m, 1 H, H-2′a), 2.05 (m, 1 H, H-2′b), 1.93 (d, 3 H, J=1.2 Hz,5-Me), 0.90 (s, 9 H, (CH₃)₃CSi), 0.09 (s, 3 H, (CH₃)Si), 0.08 (s, 3 H,(CH₃)Si).

N³-(2-Nitrobenzyl)-thymidine (dT.03)

A solution of n-Bu₄NF (1.0 M in THF, 1.125 mL, 1.125 mmol) was addeddropwise to a solution of compound dT.02 (369 mg, 0.75 mmol) in THF(3.75 mL). The reaction mixture was stirred at room temperature for 45minutes, concentrated in vacuo, and purified by silica gel columnchromatography to yield N³-(2-nitrobenzyl)-thymidine dT.03 (225 mg, 80%)as a white foam.

¹H NMR (CDCl₃): δ 8.01 (dd, 1 H, J=8.2, 1.3 Hz, Ph-H), 7.51 (m, 2 H, H-6and Ph-H), 7.40 (m, 1 H, Ph-H), 7.21 (dd, 1 H, J=7.8, 0.9 Hz, Ph-H),6.21 (t, 1 H, J=6.7 Hz, H-1′), 5.49 (dd, 2 H, PhCH₂), 4.53 (m, 1 H,H-4′), 3.97 (m, 1 H, H-3′), 3.78 (m, 2 H, H-5′a and H-5′b), 2.30 (m, 2H, H-2′a and H-2′b), 1.94 (s, 3 H, 5-CH₃).

N³-(2-Nitrobenzyl)-thymidine-5′-triphosphate (WW1p050)

POCl₃ (30 μL, 0.33 mmol) was added to a solution of compound dT.03 (38mg, 0.11 mmol) and proton sponge (32 mg, 0.15 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for six hours. Asolution of bis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added to the solution. The reaction was stirred at roomtemperature for one hour and then lyophilized to dryness. The residueobtained was dissolved in water (10 mL), filtered, and purified by anionexchange chromatography using a Q Sepharose FF column (2.5×20 cm) with alinear gradient of NH₄HCO₃ (50 mM to 500 mM in 240 minutes) at a flowrate of 4.5 mL/min. The fractions containing triphosphate were combinedand lyophilized to give triphosphateN³-(2-nitrobenzyl)-thymidine-5′-triphosphate WW1p050 (38 mg, 56%) as awhite fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.15 (d, 1 H, J=8.2 Hz, Ph-H), 7.82 (s, 1 H,H-6), 7.64 (t, 1 H, J=7.6 Hz, Ph-H), 7.54 (t, 1 H, J=7.6 Hz, Ph-H), 7.24(d, 1 H, J=7.8 Hz, Ph-H), 6.35 (t, 1 H, J=6.7 Hz, H-1′), 5.47 (s, 2 H,Ph-CH₂), 4.64 (m, 1 H, H-4′), 4.25 (m, 3 H, H-3′, H-5′a and H-5′b), 2.40(m, 2 H, H-2′a and H-2′b), 1.98 (s, 3 H, 5-CH₃);

³¹P NMR (162 MHz, D₂O): δ-6.12 (d, J=15.6 Hz), −11.21 (d, J=15.4 Hz),−19.565 (d, J=15.6 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₀N₃O₁₆P₃Na [M-2H+Na]⁻, thecalculated mass was 637.9954, and the observed mass was 637.9802.

Synthesis of 5-(2-nitrobenzyloxymethyl)-2′-deoxyuridine-5′-triphosphate(VL3p03085)

Scheme. Synthesis of5-(2-nitrobenzyloxymethyl)-2′-deoxyuridine-5′-triphosphate. (i) TBSCl,imidazole, anhydrous DMF, room temperature, overnight, 90%; (ii) NBS,benzoyl peroxide, CCl₄, reflux, one hour, 44%; (iii) 2-nitrobenzylalcohol, 110-115° C., 10 minutes, 35%; (iv) POCl₃, proton sponge,(MeO)₃PO, 0° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 MHNEt₃HCO₃, one hour, 22%.

3′,5′-O-Bis-tert-butyldimethylsilyl-thymidine (dT.04)

To a solution of thymidine (5.00 g, 20.64 mmol) and imidiazole (9.0 g,132.1 mmol) in anhydrous DMF (11 mL), a solution of TBSCl (9.96 g, 66.05mmol) in DMF (11 mL) was added dropwise, and the mixture was stirred atroom temperature overnight under a N₂ atmosphere. After the mixture wasdiluted with water (100 mL), the formed precipitate was filtered anddissolved in ethyl ether (125 mL). The ether solution was washed twicewith water (25 mL each) and once with brine (25 mL), dried over Na₂SO₄,and concentrated under reduced pressure to a waxy solid, which wasre-crystallized from hexane/ethyl either (10:1) to yield3′,5′-O-bis-tert-butyldimethylsilyl-thymidine dT.04 (10.64 g, 90%).

¹H NMR (400 MHz, CDCl₃): δ 8.51 (br s, 1 H, H-3), 7.48 (d, 1 H, J=1.2Hz, H-6), 6.34 (dd, 1 H, J=5.8 and 8.0 Hz, H-1′), 4.41 (m, 1 H, H-3′),3.93 (m, 2 H, H-4′), 3.87 (dd, 1 H, J=2.6 and 11.4 Hz, H-5′a), 3.76 (dd,1 H, J=2.6 and 11.4 Hz, H-5′b), 2.17 (m, 1 H, H-2′a), 2.01 (m, 1 H,H-2′b), 1.92 (d, 3 H, J=1.2 Hz, CH₃), 0.93 (s, 9 H, (CH₃)₃CSi), 0.88 (s,9 H, (CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si), 0.08 (s, 6 H, (CH₃)₂Si).

5-Bromomethyl-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyuridine(dT.05)

A solution of compound dT.04 (4.63 g, 9.83 mmol), N-bromosuccinimide(3.68 g, 20.68 mmol), and benzoyl peroxide (0.10 g, 75% aqueoussolution) in CCl₄ (100 mL) was refluxed for one hour. The mixture wasfiltered and the filtrate was concentrated in vacuo and purified bysilica gel column chromatography to yield5-bromomethyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyuridine dT.05(2.40 g, 44%).

¹H NMR (400 MHz, CDCl₃): δ 9.16 (br s, 1 H, H-3), 7.89 (s, 1 H, H-6),6.30 (dd, 1 H, J=5.8 and 7.7 Hz, H-1′), 4.41 (m, 1 H, H-3′), 4.29 (d, 1H, J=10.6 Hz, CH₂Br), 4.23 (d, 1 H, J=10.6 Hz, CH₂Br), 3.98 (m, 2 H,H-4′), 3.89 (dd, 1 H, J=2.6 and 11.4 Hz, H-5′b), 3.78 (dd, 1 H, J=2.6and 11.4, Hz, H-5′a), 2.30 (m, 1 H, H-2′a), 2.01 (m, 1 H, H-2′b), 0.95(s, 9 H, (CH₃)₃CSi), 0.91 (s, 9 H, (CH₃)₃CSi), 0.15 (s, 6 H, (CH₃)₂Si),0.09 (s, 6 H, (CH₃)₂Si).

5-(2-Nitrobenzyloxymethyl)-2′-deoxyuridine (dT.06)

Compound dT.05 (238 mg, 0.43 mmol) and 2-nitrobenzyl alcohol (331 mg,2.17 mmol) was heated neat at 110-115° C. for 10 minutes under a N₂atmosphere. The mixture was cooled down to room temperature, dissolvedin ethyl acetate, and purified by silica gel chromatography to yield5-(2-nitrobenzyloxymethyl)-2′-deoxyuridine dT.06 (60 mg, 35%).

¹H NMR (400 MHz, DMSO-d₆): δ 11.41 (br s, 1 H, D₂O exchangeable, NH),8.05 (d, J=8.0 Hz, 1 H, Ph-H), 7.96 (s, 1 H, H-6), 7.78 (m, 2 H, Ph-H),7.56 (m, 1 H, Ph-H), 6.17 (t, 1 H, J=6.8 Hz, H-1′), 5.25 (d, 1 H, D₂Oexchangeable, 3′-OH), 5.00 (t, 1 H, J=5.0 Hz, D₂O exchangeable, 5′-OH),4.48 (s, 2 H, CH₂), 4.24 (m, 1 H, H-3′), 4.23 (s, 2H, CH₂), 3.79 (m, 1H, H-4′), 3.57 (m, 2 H, H-5′), 2.11 (m, 2 H, H-2′);

¹³C NMR (100 MHz, MeOH-d₄): δ 163.38 (C), 147.18 (C), 139.53 (CH),134.80 (C), 133.81 (C), 132.87 (CH), 128.53 (CH), 127.63 (CH), 123.75(CH), 110.24 (C), 87.16 (CH), 82.83 (CH), 70.41 (CH), 68.26 (CH₂), 64.73(CH₂), 61.05 (CH₂), 39.58 (CH₂);

ToF-MS (ESI): For the molecular ion C₁₇H₂₀N₃O₈ [M+H]⁺, the calculatedmass was 394.1250, and the observed mass was 394.1286.

5-(2-Nitrobenzyloxymethyl)-2′-deoxyuridine-5′-triphosphate (VL3p03085)

POCl₃ (22 μL, 0.24 mmol) was added to a solution of compound dT.06 (48mg, 0.12 mmol) and proton sponge (39 mg, 0.18 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (285 mg, 0.6 mmol) andtri-n-butylamine (120 μL) in anhydrous DMF (1.2 mL) was added. After twominutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred at room temperature for onehour and then lyophilized to dryness. The residue was dissolved in water(10 mL), filtered, and purified by anion exchange chromatography using aQ Sepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50mM to 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to give5-(2-nitrobenzyloxymethyl)-2′-deoxyuridine-5′-triphosphate VL3p03085 (16mg, 22%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.01 (d, J=8.0 Hz, 1 H, Ph-H), 7.78 (s, 1 H,H-6), 7.65 (m, 2 H, Ph-H), 7.52 (m, 1 H, Ph-H), 6.26 (t, J=6.8 Hz, 1 H,H-1′), 4.93 (m, 2 H, CH₂), 4.57 (m, 1 H, H-3′), 4.41 (s, 2 H, CH₂), 4.21(m, 3 H, H-4′ and H-5′), 2.34 (m, 2 H, H-2′);

³¹P NMR (162 Hz, D₂O): δ-5.58 (d, J=18.5 Hz), −10.91 (d, J=18.5 Hz),−20.80 (br);

TOF-MS (ESI): For the molecular ion C₁₇H₂₁N₃O₁₇P₃ [M-H]⁻, the calculatedmass was 632.0084, and the observed mass was 631.9779.

Synthesis of5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p043)

Scheme. Synthesis of5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine-5′-triphosphate. (i)1-(2-nitrophenyl)ethanol (1.25 g, 7.50 mmol), 110-115° C., 10 minutes,8%; (ii) POCl₃, proton sponge, (MeO)₃PO, 0° C., three hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃, one hour, 55%.

5-[1-(2-Nitrophenyl)ethyloxymethyl]-2′-deoxyuridine (dT.07)

Compound dT.05 (0.81 g, 1.5 mmol) and 1-(2-nitrophenyl)ethanol (1.25 g,7.50 mmol) were heated neat at 110-115° C. for 10 minutes under a N₂atmosphere. The mixture was cooled down to room temperature, dissolvedin ethyl acetate, and purified by silica gel chromatography to yield5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine dT.07 (48 mg, 8%,1:1 mixture of diastereomers).

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 11.36 and 11.35 (2 br s,1 H, D₂O exchangeable, NH), 7.95 (m, 1 H, Ph-H), 7.87 (m, 1 H, Ph-H),7.76 (m, 2 H, H-6 and Ph-H), 7.54 (m, 1 H, Ph-H), 6.14 (m, 1 H, H-1′),5.25 (d, 1 H, D₂O exchangeable, 3′-OH), 5.00 (m, 2 H, among them 1 H D₂Oexchangeable, 5′-OH and CH), 4.24 (m, 1 H, H-3′), 3.96 (m, 2 H, CH₂),3.78 (m, 1 H, H-4′), 3.57 (m, 2 H, H-5′), 2.08 (m, 2 H, H-2′), 1.43 (d,J=6.3 Hz, 3 H, CH₃);

ToF-MS (ESI): For the molecular ion C₁₈H₂₂N₃O₈ [M+H]⁺, the calculatedmass was 408.1407, and the observed mass was 408.1446.

5-[1-(2-Nitrophenyl)ethyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p043)

POCl₃ (15 μL, 0.17 mmol) was added to a solution of compound dT.07 (34mg, 0.08 mmol) and proton sponge (27 mg, 0.12 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for three hours. Asolution of tri-n-butylammonium pyrophosphate (197 mg, 0.4 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (0.8 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5, 10 mL) was added. The reaction was stirred at room temperature forone hour and then lyophilized to dryness. The residue was dissolved inwater (10 mL), filtered, and purified by anion exchange chromatographyusing a Q Sepharose FF column (2.5×20 cm) with a linear gradient ofNH₄HCO₃ (50 mM to 500 mM in 300 minutes) at a flow rate of 4.5 mL/min.The fractions containing triphosphate were combined and lyophilized togive 5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine-5′-triphosphateWW2p043 (32 mg, 55%, 1:1 mixture of diastereomers) as a white fluffysolid.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 7.93 (m, 1 H, Ph-H),7.71-7.61 (m, 3H, H-6 and Ph-H), 7.49 (m, 1 H, Ph-H), 6.18 and 6.12 (2t, J=6.6 Hz, 1 H, H-1′), 5.13 (m, 1 H, CH), 4.53 (m, 1 H, H-3′), 4.39(m, 1 H, H-4′), 4.20 (m, 4 H, CH₂ and H-5′), 2.28 (m, 2 H, H-2′), 1.54(d, 3 H, J=6.3 Hz, CH₃);

³¹P NMR (162 MHz, D₂O): δ-7.98 (br), −12.64 (br), −23.33 (br);

ToF-MS (ESI): For the molecular ion C₁₈H₂₄N₃O₁₇P₃Na [M+Na]⁺, thecalculated mass was 670.0216, and the observed mass was 670.0176.

Synthesis of 6-JOE labeled5-[4-(3-amino-1-propynyl)-2-nitrobenzyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p075)

Scheme. Synthesis of 6-JOE labeled5-[4-(3-amino-1-propynyl)-2-nitrobenzyl-oxymethyl]-2′-deoxyuridine-5′-triphosphate.(i) 4-iodo-2-nitrobenzyl alcohol, neat, 110-115° C., 10 minutes, 10%;(ii) N-propargyltrifluoroacetamide, Pd(PPh₃)₄ (O), CuI, Et₃N, anhydrousDMF, four hours, 50%; (iii) POCl₃, proton sponge, (MeO)₃PO, 0° C., fourhours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, onehour; NH₄OH, one hour; 31%; (iv) 6-JOE-SE, 0.1 M Na₂CO₃/NaHCO₃ buffer(pH 9.2), one hour.

5-(4-Iodo-2-nitrobenzyloxymethyl)-2′-deoxyuridine (dT.08)

Compound dT.05 (0.59 g, 1.06 mmol) and 4-iodo-2-nitrobenzyl alcohol(1.09 g, 3.9 mmol) were heated neat at 110-115° C. for 10 minutes undera N₂ atmosphere. The mixture was cooled down to room temperature,dissolved in ethyl acetate, and purified by silica gel chromatography toyield 5-(4-iodo-2-nitrobenzyloxymethyl)-2′-deoxyuridine dT.08 (52 mg,10%) as a low-melting solid.

¹H NMR (400 MHz, DMSO-d₆): δ 11.42 (s, 1 H, D₂O exchangeable, N—H), 8.34(d, 1 H, J=1.7 Hz, Ph-H), 8.09 (dd, 1 H, J=1.7 and 8.2 Hz, Ph-H), 7.96(s, 1 H, H-6), 7.56 (d, 1 H, J=8.2, Ph-H), 6.16 (t, 1 H, J=6.8 Hz,H-1′), 5.25 (d, 1 H, D₂O exchangeable, 3′-OH), 5.02 (t, 1 H, D₂Oexchangeable, 5′-OH), 4.77 (s, 2 H, CH₂), 4.20 (m, 1 H, H-3′), 4.22 (s,2 H, CH₂), 3.79 (m, 1 H, H-4′), 3.57 (m, 2 H, H-5′), 2.11 (m, 2 H,H-2′).

5-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyloxymethyl]-2′-deoxyuridine(dT.09)

A solution of compound dT.08 (51 mg, 0.1 mmol),N-propargyltrifluoroacetylamide (45 mg, 0.3 mmol),tetrakis(triphenylphosphine)-palladium(0) (12 mg, 0.01 mmol), CuI (4 mg,0.02 mmol), and Et₃N (28 μL, 0.2 mmol) in anhydrous DMF (1.2 mL) wasstirred at room temperature for four hours. The mixture was concentratedin vacuo and purified by silica gel column chromatography to yield5-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyloxymethyl]-2′-deoxyuridinedT.09 (27 mg, 50%) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆): δ 11.44 (s, 1 H, D₂O exchangeable, N3-H),10.44 (1 H, D₂O exchangeable, N—H(COCF₃)), 8.06 (s, 1 H, Ph-H), 7.97 (s,1 H, H-6), 7.79 (s, 2 H, Ph-H), 6.16 (t, J=6.8 Hz, 1 H, H-1′), 5.25 (d,1 H, D₂O exchangeable, 3′-OH), 5.02 (t, 1 H, D₂O exchangeable, 5′-OH),4.83 (s, 2 H, Ph-CH₂), 4.30 (s, 2 H, CH₂), 4.23 (m, 3H, CH₂ and H-3′),3.79 (m, 1 H, H-4′a), 3.57 (m, 2 H, H-5′), 2.10 (m, 2 H, H-2′a andH-2′b);

ES+ MS (ESI): 543 [M+H]⁺; ES− MS (ESI): 541 [M+H]⁺

5-[4-(3-Amino-1-propynyl)-2-nitrobenzyloxymethyl]-2′-deoxyuridine-5′-triphosphate(dT.10)

POCl₃ (8 μL, 88 μmol) was added to a solution of compound dT.09 (24 mg,44 μmol) and proton sponge (14 mg, 66 μmol) in trimethylphosphate (0.5mL) at 0° C. and stirred for two hours. Additional POCl₃ (8 μL, 88 μmol)was added and stirred for another two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (104 mg, 0.22 mmol) andtri-n-butylamine (50 μL) in anhydrous DMF (0.5 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred at room temperature for onehour, followed by the dropwise addition of concentrated ammoniumhydroxide (5 mL, 27%) at 0° C. The mixture was stirred for an additionalhour at room temperature and then lyophilized to dryness. The residuewas dissolved in water (10 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×20 cm) with a lineargradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes) at a flow rate of4.5 mL/min. The fractions containing triphosphate were combined andlyophilized to give triphosphate dT.10 (10 mg, 31%) as a white fluffysolid.

¹H NMR (400 MHz, D₂O): δ 8.10 (d, J=5.1 Hz, 1 H, Ph-H), 7.75 (m, 2 H,H-6 and Ph-H), 7.65 (m, 1 H, J=8.0 Hz, Ph-H), 6.27 (t, J=6.8 Hz, 1 H,H-1′), 4.95 (m, 2 H, CH₂), 4.58 (m, 1 H, H-3′), 4.43 (s, 2 H, CH₂), 4.22(m, 3 H, H-4′ and H-5′), 3.64 (s, 2 H, CH₂), 2.33 (m, 2 H, H-2′);

³¹P NMR (162 Hz, D₂O): δ-6.51 (d, J=15.0 Hz), −11.56 (d, J=15.6 Hz),−19.82 (t, J=15.0 Hz);

TOF-MS (ESI): For the molecular ion C₂₀H₂₃N₄O₁₇P₃Na [M-2H+Na]⁻, thecalculated mass was 707.0169, and the observed mass was 707.0321.

6-JOE Labeled5-[4-(3-Amino-1-propynyl)-2-nitrobenzyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p075)

A solution of 6-JOE-SE (1.25 mg, 2 μmol) in anhydrous DMSO (50 μL) wasadded to a solution of triphosphate dT.10 (1.4 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 0.5 mL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-JOE labeledtriphosphate WW2p075. Mobile phase: A, 100 mM triethylammonium acetate(TEAA) in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). Elutionwas performed with a linear gradient of 5-38% B for 40 minutes and then38-90% B for 10 minutes. The concentration of WW2p075 was estimated byadsorption spectroscopy using the extinction coefficient of the 6-JOEdye (i.e., 75,000 at 520 nm).

Synthesis of 6-JOE labeled5-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl-oxymethyl}-2′-deoxyuridine-5′-triphosphate(WW2p113)

Scheme. Synthesis of 6-JOE labeled5-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-ethyloxy-methyl}-2′-deoxyuridine-5′-triphosphate.(i) Boc₂O, DMAP, anhydrous DMF, room temperature, 16 hours, 78%; (ii)NBS, benzoyl peroxide, CCl₄, reflux, one hour, 43%; (iii)1-(4-iodo-2-nitrophenyl)ethanol, neat, 95° C., 50 minutes, 13%; (iv)N-propargyltrifluoroacetamide, Pd(PPh₃)₄ (0), CuI, Et₃N, anhydrous DMF,four hours, 76%; (v) POCl₃, proton sponge, (MeO)₃PO, 0° C.;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃, one hour; NH₄OH, one hour;31%; (vi) 6-JOE-SE, 0.1 M Na₂CO₃/NaHCO₃ buffer (pH 9.2), one hour.

N³-tert-Butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-thymidine(dT.11)

To a solution of compound dT.04 (2.43 g, 5.15 mmol) and DMAP (1.39 g,11.34 mmol) in anhydrous DMF (45 mL), a solution ofdi-tert-butyldicarbonate (2.47 g, 11.34 mmol) in DMF (9 mL) was addeddropwise. The mixture was stirred at room temperature for 16 hours undera N₂ atmosphere. The mixture was concentrated in vacuo, and thecrystalline residue was dissolved in CH₂Cl₂ (80 mL), washed withsaturated NH₄Cl solution (10 mL), dried over Na₂SO₄, and concentrated invacuo. The residue was purified by silica gel column chromatography toyieldN³-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-thymidinedT.11 (2.30 g, 78%) as a white solid.

¹H NMR (400 MHz, CDCl₃): δ 7.50 (d, 1 H, J=1.1 Hz, H-6), 6.34 (dd, 1 H,J=5.8 and 7.9 Hz, H-1′), 4.42 (m, 1 H, H-3′), 3.95 (m, 2 H, H-4′), 3.87(dd, 1 H, J=2.5 and 11.4 Hz, H-5′a), 3.76 (dd, 1 H, J=2.5 and 11.4 Hz,H-5′b), 2.17 (m, 1 H, H-2′a), 2.01 (m, 1 H, H-2′b), 1.92 (d, 3 H, J=1.2Hz, CH₃), 1.60 (s, 9 H, (CH₃)₃COCON), 0.93 (s, 9 H, (CH₃)₃CSi), 0.88 (s,9 H, (CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si), 0.08 (s, 6 H, (CH₃)₂Si).

N³-tert-Butyloxycarbonyl-5-bromomethyl-3′,5′-bis-O-tert-butyldimethylsilyl-2′-deoxyuridine(dT.12)

A solution of compound dT.11 (570 mg, 1.00 mmol), N-bromosuccinimide(0.37 g, 2.10 mmol), and benzoyl peroxide (10 mg, 75% aqueous solution)in CCl₄ (10 mL) was refluxed for one hour. The mixture was filtered,concentrated in vacuo, and purified by silica gel column chromatographyto yieldN³-tert-butyloxycarbonyl-5-bromomethyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyuridinedT.12 (281 mg, 43%) as a waxy solid.

¹H NMR (400 MHz, CDCl₃): δ 7.89 (s, 1 H, H-6), 6.27 (dd, 1 H, J=5.7 and7.7 Hz, H-1′), 4.39 (m, 1 H, H-3′), 4.27 (d, 1 H, J=10.6 Hz, CH₂Br),4.20 (d, 1 H, J=10.6 Hz, CH₂Br), 3.98 (m, 2 H, H-4′), 3.89 (dd, 1 H,J=2.5 and 11.4, Hz, H-5′b), 3.78 (dd, 1 H, J=2.6 and 11.4 Hz, H-5′a),2.30 (m, 1 H, H-2′a), 2.04 (m, 1 H, H-2′b), 1.61 (s, 9 H, (CH₃)₃COCON),0.95 (s, 9 H, (CH₃)₃CSi), 0.89 (s, 9 H, (CH₃)₃CSi), 0.14 (s, 6 H,(CH₃)₂Si), 0.07 (s, 6 H, (CH₃)₂Si);

¹³C NMR (100 MHz, CDCl₃): δ 159.21 (C), 147.99 (C), 147.32 (C), 138.46(CH), 111.30 (C), 88.34 (CH), 87.22 (C), 86.00 (CH), 72.28 (CH), 63.04(CH₂), 41.93 (CH₂), 27.42 (CH₃), 25.99 (CH₃), 25.71 (CH₃), 25.65 (CH₃),24.91 (CH₂), 18.47 (C), 17.97 (C), −3.58 (CH₃), −4.65 (CH₃), −4.86(CH₃), −5.32 (CH₃).

5-[1-(4-Iodo-2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine (dT.13)

Compound dT.12 (323 mg, 0.50 mmol) and 1-(4-iodo-2-nitrophenyl)ethanol(293 mg, 2.23 mmol) were heated neat at 95-97° C. for 50 minutes under aN₂ atmosphere. The mixture was cooled down to room temperature,dissolved in ethyl acetate, and purified by silica gel chromatography toyield 5-[1-(4-iodo-2-nitrophenyl)ethyloxymethyl]-2′-deoxyuridine dT.13(34 mg, 13%, 1:1 mixture of diastereomers) as a waxy solid.

¹H NMR (400 MHz, MeOH-d₄) for diastereomers: δ 8.26 (t, 1 H, J=1.7 Hz,H-6), 8.05 (dd, 1 H, J=1.6, 8.3 Hz, Ph-H), 8.02 (d, 1 H, J=10.6 Hz,Ph-H), 7.60 (dd, 1 H, J=1.1, 8.3 Hz, Ph-H), 6.26 (m, 1 H, H-1′), 5.03(m, 1 H, PhCH), 4.41 (m, 1 H, H-3′), 4.10 (m, 2 H, CH₂), 3.94 (m, 1 H,H-4′), 3.80 (m, 1 H, H-5′a), 3.74 (m, 1 H, H-5′b), 2.30 (m, 1 H, H-2′a),2.20 (m, 1 H, H-2′b), 1.50 (m, 3 H, CH₃).

5-{1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyloxymethyl}-2′-deoxyuridine(dT.14)

A solution of compound dT.13 (52 mg, 0.1 mmol),N-propargyltrifluoroacetylamide (44 mg, 0.29 mmol),tetrakis(triphenylphosphine)-palladium(0) (12 mg, 0.01 mmol), CuI (4 mg,0.02 mmol), and Et₃N (27 μL, 0.2 mmol) in anhydrous DMF (1.5 mL) wasstirred at room temperature for four hours. The mixture was concentratedin vacuo and purified by silica gel column chromatography to yield5-[1-(4-{3-trifluoroacetamido-1-propynyl}-2-nitrophenyl)ethyloxymethyl]-2′-deoxy-uridinedT.14 (41 mg, 76%, 1:1 mixture of diastereomers) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 11.36, 11.35(2 s, 1 H,D₂O exchangeable, NH), 10.11 (t, 1 H, J=5.6 Hz, D₂O exchangeable, NH),7.98 (s, 1 H, H-6), 7.88 (d, 1 H, J=8.1 Hz, Ph-H), 7.78 (m, 2 H, Ph-H),6.14 (t, J=7.0 Hz, 1 H, H-1′), 5.25 (m, 1 H, D₂O exchangeable, 3′-OH),5.01 (m, 1 H, D₂O exchangeable, 5′-OH), 4.98 (m, 1 H, PhCH), 4.33 (m, 2H, CH₂), 4.24 (m, 1 H, H-3′), 4.00 (m, 1 H, H-5′a), 3.94 (m, 1 H,H-5′b), 3.78 (m, 1 H, H-4′), 3.57 (m, 2 H, CH₂), 2.08 (m, 2 H, H-2′a andH-2′b), 1.41 (d, J=8.1 Hz, 3 H, CH₃);

ES⁺ MS (ESI): 579 [M+Na]⁺.

5-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]ethyloxymethyl}-2′-deoxyuridine-5′-triphosphate(dT.15)

POCl₃ (10 μL, 0.11 mmol) was added to a solution of compound dT.14 (30mg, 0.054 mmol) and proton sponge (17 mg, 0.08 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for two hours.Additional POCl₃ (2.5 μL, 0.03 mmol) was added and stirred for anotherhour. A solution of bis-tri-n-butylammonium pyrophosphate (128 mg, 0.27mmol) and tri-n-butylamine (60 μL) in anhydrous DMF (0.54 mL) was added.After five minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred at roomtemperature for one hour, followed by the dropwise addition ofconcentrated ammonium hydroxide (5 mL, 27%) at 0° C. The mixture wasstirred at room temperature for an additional hour and then lyophilizedto dryness. The residue was dissolved in water (10 mL), filtered, andpurified by anion exchange chromatography using a Q Sepharose FF column(2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mM to 500 mM in 300minutes) at a flow rate of 4.5 mL/min. The fractions containingtriphosphate were combined and lyophilized to give triphosphate dT.15(16 mg, 40%, 1:1 mixture of diastereomers) as a white fluffy solid.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 8.01 (m, 1 H, Ph-H),7.74-7.55 (m, 3H, H-6 and Ph-H), 6.18 and 6.12 (2 t, J=6.4 Hz, 1 H,H-1′), 5.11 (m, 1 H, PhCH), 4.53 (m, 1 H, H-3′), 4.37 (m, 1 H, H-4′),4.20 (m, 4 H, CH₂ and H-5′), 3.65 (s, 2 H, CH₂), 2.35 (m, 1 H, H-2′a),2.25 (m, 1 H, H-2′b), 1.54 (d, 3 H, J=6.4 Hz, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: δ-5.87 (d, J=19.8 Hz), −11.18and −11.30 (2 d, J=19.4 Hz), −21.62 (t, J=19.6 Hz);

ToF-MS (ESI): For the molecular ion C₂₁H₂₇N₄O₁₇P₃Na [M+Na]⁺, thecalculated mass was 723.0482, and the observed mass was 723.0497.

6-JOE Labeled5-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]ethyloxymethyl}-2′-deoxy-uridine-5′-triphosphate(WW2p113)

A solution of 6-JOE-SE (0.75 mg, 1.2 μmol) in anhydrous DMSO (30 μL) wasadded to a solution of triphosphate dT.15 (0.56 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 200 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-JOE labeledtriphosphate WW2p113. Mobile phase: A, 100 mM TEAA in water (pH 7.0); B,100 mM TEAA in water/CH₃CN (30:70). Elution was performed with a lineargradient of 5-50% B for 40 minutes and then 50-90% B for 10 minutes. Theconcentration of WW2p113 was estimated by adsorption spectroscopy usingthe extinction coefficient of the 6-JOE dye (i.e., 75,000 at 520 nm).

Synthesis of5-[1-(2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p148)

Scheme. Synthesis of5-[1-(2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine-5′-triphosphate.(i) 1-(2-nitrophenyl)-2-methylpropanol, 100-110° C., 35 minutes, 14%;(ii) POCl₃, proton sponge, (MeO)₃PO, 0° C., three hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 M HNEt₃HCO₃, one hour.

5-[1-(2-Nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine (dT.16)

Compound dT.12 (0.316 g, 0.486 mmol) and1-(2-nitrophenyl)-2-methylpropanol (0.706 g, 3.62 mmol) were heated neatat 100-110° C. for 35 minutes under a nitrogen atmosphere. The mixturewas cooled down to room temperature, dissolved in ethyl acetate, andpurified by silica gel chromatography to yield5-[1-(2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine dT.16(30 mg, 14%, 1:1 mixture of diastereomers).

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 11.33 and 11.34 (2 br s,1 H, D₂O exchangeable, NH), 7.95 (m, 1 H, Ph-H), 7.83 (m, 1 H, Ph-H),7.72 (m, 1 H, Ph-H), 7.68 (m, 1 H, H-6), 7.54 (m, 1 H, Ph-H), 6.14 (m, 1H, H-1′), 5.26 (d, 1 H, D₂O exchangeable, 3′-OH), 4.97 (m, 1 H, 1 H D₂Oexchangeable, 5′-OH), 4.66 (m, 1 H, CH), 4.22 (m, 1 H, H-3′), 3.98 (m, 2H, CH₂), 3.78 (m, 1 H, H-4′), 3.55 (m, 2 H, H-5′), 2.08 (m, 2 H, H-2′),1.72 (m, 1 H, CH), 0.85 (m, 3 H, CH₃), 0.80 (m, 3 H, CH₃);

¹³C NMR (100 MHz, CDCl₃) for diastereomers: δ 165.04 (C), 152.20/151.09(C), 141.19/141.04 (CH), 139.28 (C), 138.05 (C), 134.08 (CH),130.57/130.51 (CH), 129.60 (CH), 125.25/125.19 (CH), 112.62/112.43 (C),89.12 (CH)/86.64 (CH), 82.72/82.40 (CH), 72.45 (CH), 65.78/65.61 (CH₂),63.01 (CH₂), 41.50/41.45 (CH₂), 36.21 (CH), 26.68 (CH), 19.85/19.80(CH₃), 18.20/18.14 (CH₃);

5-[1-(2-Nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine-5′-triphosphate(WW2p148)

POCl₃ (13 μL, 0.17 mmol) was added to a solution of compound dT.16 (30mg, 0.07 mmol) and proton sponge (30 mg, 0.14 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for three hours. Asolution of tri-n-butylammonium pyrophosphate (166 mg, 0.35 mmol) andtri-n-butylamine (70 μL) in anhydrous DMF (0.7 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred at room temperature for onehour and then lyophilized to dryness. The residue was dissolved in water(10 mL), filtered, and part of the solution was purified by anionexchange chromatography using a Q Sepharose FF column (2.5×20 cm) with alinear gradient of NH₄HCO₃ (50 mM to 500 mM in 240 minutes) at a flowrate of 4.5 mL/min. The fractions containing triphosphate were combinedand lyophilized to give5-[1-(2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine-5′-triphosphateWW2p148 (1:1 mixture of diastereomers) as a white fluffy solid.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 7.93 (m, 1 H, Ph-H),7.74-7.64 (m, 3 H, H-6 and Ph-H), 7.52 (m, 1 H, Ph-H), 6.19 and 6.13 (2t, J=6.6 Hz, 1 H, H-1′), 4.55 (m, 1 H, H-3′), 4.40 (m, 1 H, H-4′), 4.21(m, 4 H, CH₂ and H-5′), 2.38-2.22 (m, 2 H, H-2′), 1.99 (m, 1 H, CH),1.01 (m, 3 H, CH₃), 0.78 (m, 3 H, CH₃).

³¹P NMR (162 MHz, D₂O) for diastereomers: δ-5.26 (d, J=20.1 Hz), −10.66and −10.72 (2 d, J=19.6 Hz), −21.17 (t, J=19.6 Hz).

ToF-MS (ESI): For the molecular ion C₂₀H₂₇N₃O₁₇P₃ [M-H]⁻, the calculatedmass was 674.0553, and the observed mass was 674.0470.

Synthesis of 6-JOE Labeled5-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(WW2p150)

Scheme. Synthesis of 6-JOE labeled5-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate.(i) 1-(4-iodo-2-nitrophenyl)-2-methylpropanol, neat, 108° C., 45minutes, 18%; (ii) N-propargyltrifluoroacetamide, Pd(PPh₃)₄ (O), CuI,Et₃N, anhydrous DMF, 4.5 hours, 99%; (iii) POCl₃, proton sponge,(MeO)₃PO, 0° C., three hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 MHNEt₃HCO₃, one hour; NH₄OH, one hour; (iv) 6-JOE-SE, 0.1 M Na₂CO₃/NaHCO₃buffer (pH 9.2), one hour.

5-[1-(4-Iodo-2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine(dT.17)

Compound dT.12 (400 mg, 0.615 mmol) and1-(4-iodo-2-nitrophenyl)-2-methylethanol (800 mg, 2.49 mmol) were heatedneat at 108° C. for 45 minutes under a nitrogen atmosphere. The mixturewas cooled down to room temperature, dissolved in ethyl acetate, andpurified by silica gel chromatography to yield5-[1-(4-iodo-2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridinedT.17 (64 mg, 18%, 1:1 mixture of diastereomers) as a waxy solid.

¹H NMR (400 MHz, MeOH-d₄) for diastereomers: δ 8.22 (m, 1 H, H-6), 8.02(m, 2 H, Ph-H), 7.49 (m, 1 H, Ph-H), 6.22 (m, 1 H, H-1′), 4.69 (m, 1 H,CH), 4.41 (m, 1 H, H-3′), 4.10 (m, 2 H, CH₂), 3.92 (m, 1 H, H-4′), 3.75(m, 2 H, H-5′a), 2.17 (m, 1 H, H-2′a), 2.15 (m, 1 H, H-2′b), 1.90(m, 1H, CH), 0.92 (m, 3 H, CH₃), 0.85 (m, 3 H, CH₃);

¹³C NMR (100 MHz, CDCl₃) for diastereomers: δ 165.11 (C), 152.16/151.18(C), 143.12 (CH), 141.44/141.32 (CH), 137.91/137.88 (C), 133.83/133.77(CH), 132.37/132.33 (CH), 130.80/129.67 (C), 112.40/112.24 (C), 92.75(C), 89.12 (CH)/86.90 (CH), 82.43/82.18 (CH), 72.39/72.37 (CH),65.83/65.70 (CH₂), 62.96 (CH₂), 41.56/41.49 (CH₂), 36.01 (CH),27.83/26.37 (CH), 19.82/19.78 (CH₃), 17.91/17.88 (CH₃);

5-{1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-(methyl)]propyloxymethyl}-2′-deoxyuridine(dT.18)

A solution of compound dT.17 (60 mg, 0.107 mmol),N-propargyl-trifluoroacetylamide (48.5 mg, 0.321 mmol),tetrakis(triphenylphosphine)-palladium(0) (12.4 mg, 0.01 mmol), CuI (4mg, 0.02 mmol), and Et₃N (30 μL, 0.214 mmol) in anhydrous DMF (1.5 mL)was stirred at room temperature for 4.5 hours. The mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield5-[1-(4-{3-trifluoroacetamido-1-propynyl}-2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridinedT.18 (62 mg, 99%, 1:1 mixture of diastereomers) as a waxy solid.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 11.36, 11.35(2 s, 1 H,D₂O exchangeable, N³—H), 10.11 (t, 1 H, J=5.3 Hz, D₂O exchangeable,NHTFA), 7.99 (m, 1 H, H-6), 7.86 (m, 1 H, Ph-H), 7.75 (m, 1 H, Ph-H),7.65 (m, 1 H, Ph-H), 6.12 (m, 1 H, H-1′), 5.26 (m, 1 H, D₂Oexchangeable, 3′-OH), 4.97 (m, 1 H, D₂O exchangeable, 5′-OH), 4.62 (m, 1H, CH), 4.31 (m, 2 H, CH₂), 4.30 (m, 1 H, H-3′), 3.98 (m, 2 H, CH₂),3.78 (m, 1 H, H-4′), 3.55 (m, 2 H, H-5′a and H-5′b), 2.08 (m, 2 H, H-2′aand H-2′b), 1.77 (m, 1 H, CH), 0.82 (m, 6 H, 2 CH₃);

5-{1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(dT.19)

POCl₃ (8 μL, 0.09 mmol) was added to a solution of compound dT.18 (34mg, 0.06 mmol) and proton sponge (26 mg, 0.12 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours.Additional POCl₃ (4 μL, 0.045 mmol) was added and stirred for anotherhour. A solution of bis-tri-n-butylammonium pyrophosphate (142 mg, 0.3mmol) and tri-n-butylamine (60 μL) in anhydrous DMF (0.6 mL) was added.After five minutes of stirring, triethylammonium bicarbonate buffer (1M, pH 7.5; 10 mL) was added. The reaction was stirred at roomtemperature for one hour, followed by the dropwise addition ofconcentrated ammonium hydroxide (5 mL, 27%) at 0° C. The mixture wasstirred at room temperature for an additional hour and then lyophilizedto dryness. The residue was dissolved in water (10 mL), filtered, andpart of the solution was purified by anion exchange chromatography usinga Q Sepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃(50 mM to 500 mM in 240 minutes) at a flow rate of 4.5 mL/min. Thefractions containing triphosphate were combined and lyophilized to givetriphosphate dT.19 (1:1 mixture of diastereomers) as a white fluffysolid.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 8.06 and 8.04 (2 s, 1 H,Ph-H), 7.78 (m, 1 H, Ph-H), 7.69-7.59 (m, 2 H, H-6 and Ph-H), 6.13 (m, 1H, H-1′), 4.55 (m, 1 H, H-3′), 4.46 and 4.34 (2 d, 2 H, CH₂), 4.20 (m, 3H, H-4′ and H-5′), 3.87 and 3.83 (2 s, 2H, CH₂), 2.40-2.20 (m, 2 H,H-2′), 1.99 (m, 1 H, CH), 1.02 (m, 3 H, CH₃), 0.79 (m, 3 H, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: δ-5.15 (d, J=19.8 Hz), −10.48and −10.54 (2 d, J=19.6 Hz), −21.0 (m);

ToF-MS (ESI): For the molecular ion C₂₃H₃₀N₄O₁₇P₃ [M-H]⁻, the calculatedmass was 727.0819, and the observed mass was 727.0828.

6-JOE Labeled5-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(WW2p150)

A solution of 6-JOE-SE (0.75 mg, 1.2 μmol) in anhydrous DMSO (30 μL) wasadded to a solution of triphosphate WW2p145 (0.47 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 0.3 mL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-JOE labeledtriphosphate WW2p150. Mobile phase: A, 100 mM TEAA in water (pH 7.0); B,100 mM TEAA in water/CH₃CN (30:70). HPLC purification was achieved usinga linear gradient of 5-50% B for 40 minutes and then 50-90% B for 10minutes. The concentration of WW2p150 was estimated by adsorptionspectroscopy using the extinction coefficient of the 6-JOE dye (i.e.,75,000 at 520 nm).

Synthesis of 6-JOE Labeled 5-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(WW3p024)

Scheme. Synthesis of 6-JOE labeled 5-{(R orS)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate.(i) (S or R)-1-(4-iodo-2-nitrophenyl)-2-methylpropanol, neat, 108° C.,45 minutes, 20%; (ii) N-propargyltrifluoroacetamide, Pd(PPh₃)₄ (O), CuI,Et₃N, anhydrous DMF, 4.5 hours, 81%; (iii) POCl₃, proton sponge,(MeO)₃PO, 0° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF; 1 MHNEt₃HCO₃, one hour; NH₄OH, two hours; (iv) 6-JOE-SE, 0.1 MNa₂CO₃/NaHCO₃ buffer (pH 9.2), one hour.

5-[(R orS)-1-(4-Iodo-2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridine(dT.20)

Compound dT.12 (143 mg, 0.22 mmol) and enantio-pure (S orR)-1-(4-iodo-2-nitrophenyl)-2-methylpropanol (282 mg, 0.88 mmol,absolute configuration not determined) were heated neat at 108° C. for45 minutes under a nitrogen atmosphere. The mixture was cooled down toroom temperature, dissolved in ethyl acetate, and purified by silica gelchromatography to yield 5-[(R orS)-1-(4-iodo-2-nitrophenyl)-2-(methyl)propyloxymethyl]-2′-deoxyuridinedT.20 (25 mg, 20%, absolute configuration not determined) as a waxysolid.

¹H NMR (400 MHz, MeOH-d₄) δ 8.22 (d, 1 H, J=1.8 Hz, H-6), 8.01 (m, 2 H,Ph-H), 7.50 (d, 1 H, J=8.3 Hz, Ph-H), 6.25 (t, 1 H, J=7.2 Hz, H-1′),4.69 (d, 1 H, J=5.8 Hz, PhCH), 4.41 (m, 1 H, H-3′), 4.10 (m, 2 H, CH₂),3.92 (m, 1 H, H-4′), 3.75 (m, 2 H, H-5′a), 2.17 (m, 1 H, H-2′a), 2.15(m, 1 H, H-2′b), 1.90(m, 1 H, CH), 0.92 (m, 3 H, CH₃), 0.85 (m, 3 H,CH₃);

5-{(R orS)-1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-(methyl)-propyloxymethyl}-2′-deoxyuridine(dT.21)

A solution of compound dT.20 (24 mg, 0.043 mmol),N-propargyl-trifluoroacetylamide (28 mg, 0.186 mmol),tetrakis(triphenylphosphine)-palladium(0) (7.2 mg, 0.006 mmol), CuI (2.4mg, 0.012 mmol), and Et₃N (17 μL, 0.124 mmol) in anhydrous DMF (1.5 mL)was stirred at room temperature for 4.5 hours. The mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield 5-{(R orS)-1-[4-(3-trifluoro-acetamido-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl]-2′-deoxyuridinedT.21 (19.8 mg, 81%, absolute configuration not determined) as a waxysolid.

¹H NMR (400 MHz, MeOH-d₄): δ 8.01 (br s, 1 H, H-6), 7.95 (d, 1 H, J=1.2Hz, Ph-H), 7.72 (m, 2 H, Ph-H), 6.25 (t, 1 H, J=6.7 Hz, H-1′), 4.74 (d,1 H, J=5.8 Hz, PhCH), 4.38 (m, 1 H, H-3′), 4.34 (s, 2 H, CH₂), 4.05 (m,2 H, CH₂), 3.55, 3.93 (m, 1 H, H-4′), 3.77 (m, 2 H, H-5′a and H-5′b),2.15 (m, 2 H, H-2′a and H-2′b), 1.90 (m, 1 H, CH), 0.92 (m, 6 H, 2×CH₃);

5-{(R orS)-1-[4-(3-Amino-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(dT.22)

POCl₃ (6 μL, 0.06 mmol) was added to a solution of compound dT.21 (18mg, 0.03 mmol) and proton sponge (13 mg, 0.06 mmol) intrimethylphosphate (0.3 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (73 mg, 0.15 mmol) andtri-n-butylamine (30 μL) in anhydrous DMF (0.3 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 5mL) was added. The reaction was stirred for one hour at room temperatureand then lyophilized to dryness. The residue was dissolved in water (5mL), filtered, and part of the solution was purified with reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield 5-{(RorS)-1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]-2-(methyl)propyloxymethyl}-2′-deoxyuridine-5′trihosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved using a linear gradient of 5-50% B for 40 minutes and then50-90% B for 10 minutes. The purified triphosphate was then treated withconcentrated ammonium hydroxide (27%; 0.5 mL) at room temperature fortwo hours to yield 5-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-(methyl)-propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(dT.22, absolute configuration not determined).

¹H NMR (400 MHz, D₂O): δ 8.01 (s, 1 H, Ph-H), 7.76 (d, 1 H, J=6.9 Hz,Ph-H), 7.62 (m, 2 H, H-6 and Ph-H), 6.17 (t, 1 H, J=6.4 Hz, H-1′), 4.55(m, 1 H, H-3′), 4.39 and 4.29 (2 d, 2 H, J=6.4 Hz, CH₂), 4.17 (m, 3 H,H-4′ and H-5′), 3.74 (s, 2 H, CH₂), 2.28 (m, 2 H, H-2′), 2.00 (m, 1 H,CH), 0.79 (m, 3 H, CH₃);

³¹P NMR (162 MHz, D₂O): δ-5.40 (d, J=19.4 Hz), −10.75 (d, J=19.4 Hz),−21.23 (t, J=19.4 Hz).

6-JOE Labeled 5-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]-2-(methyl)-propyloxymethyl}-2′-deoxyuridine-5′-triphosphate(WW3p024)

A solution of 6-JOE-SE (0.625 mg, 1 μmol) in anhydrous DMSO (25 μL) wasadded to a solution of triphosphate dT.22 (0.31 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 180 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-JOE labeledtriphosphate WW3p024. Mobile phase: A, 100 mM TEAA in water (pH 7.0); B,100 mM TEAA in water/CH₃CN (30:70). HPLC purification was achieved usinga linear gradient of 5-50% for 40 minutes and then 50-90% B for 10minutes. The concentration of WW3p024 was estimated by adsorptionspectroscopy using the extinction coefficient of the 6-JOE dye (i.e.,75,000 at 520 nm).

Example 3 dC Compounds Synthesis ofN4-(2-nitrobenzyl)-2′-deoxycytidine-5′-triphosphate (WW2p044)

Scheme. Synthesis of N⁴-(2-nitrobenzyl)-2′-deoxycytidine triphosphate.(i) TBSCl, imidazole, anhydrous DMF, room temperature, overnight, 84%;(ii) Boc₂O, DMAP, Et₃N, CH₂Cl₂, room temperature, overnight, 56%; (iii)2-nitrobenzyl bromide, NaH, anhydrous DMF, 0° C., then gradually warmedto room temperature, overnight, 37%; (iv) SiO₂, vacuum, 70-80° C., 48hours, 79%; (v) n-Bu₄NF, THF, 0° C., then gradually warmed to roomtemperature, two hours, 59%; (vi) POCl₃, proton sponge, (MeO)₃PO, 0° C.,two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃,one hour, 52%.

3′,5′-O-Bis-tert-butyldimethylsilyl-2′-deoxycytidine (dC.01)

2′-deoxycytidine dC (2.85 g, 12.54 mmol), imidiazole (6.49 g, 95.31mmol), and TBSCl (7.18 g, 47.65 mmol) were added to anhydrous DMF (27mL) and stirred at room temperature overnight under a N₂ atmosphere.Methanol (20 mL) was added, and the mixture was stirred for 30 minutesand then concentrated in vacuo. Ethyl acetate (60 mL) and water (60 mL)were then added. The organic layer was separated and washed twice withwater (20 mL), and the combined aqueous layer was extracted with ethylacetate (20 mL). The combined organic layer was dried with Na₂SO₄,concentrated in vacuo, and purified by silica gel chromatography to give3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine dC.01 (4.79 g, 84%)as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 7.95 (d, 1 H, J=7.4 Hz, H-6), 6.26 (t, 1 H,J=5.6 Hz, H-1′), 5.69 (d, 1 H, J=7.4 Hz, H-5), 4.37 (m, 1 H, H-3′), 3.90(m, 2 H, H-4′ and H-5′a), 3.76 (m, 1 H, H-5′b), 2.40 (m, 1 H, H-2′a),2.08 (m, 1 H, H-2′b), 0.91 (s, 9 H, (CH₃)₃CSi), 0.87 (s, 9 H,(CH₃)₃CSi), 0.10 (s, 6 H, (CH₃)₂Si), 0.05 (s, 6 H, (CH₃)₂Si).

N⁴-tert-Butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.02)

Under a nitrogen atmosphere, a solution of di-tert-butyldicarbonate(0.34 g, 1.58 mmol) in anhydrous CH₂Cl₂ (3 mL) was slowly added to asolution of compound dC.01 (0.5 g, 1.10 mmol), Et₃N (0.15 mL, 1.10mmol), and DMAP (0.13 g, 1.10 mmol) in anhydrous CH₂Cl₂ (5 mL). Themixture was stirred overnight at room temperature, concentrated invacuo, and purified by silica gel chromatography to yieldN⁴-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.02 (0.34 g, 56%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.30 (d, 1 H, J=7.4 Hz H-6), 7.47 (bs, 1 H,NH), 7.14 (d, 1 H, J=7.4 Hz, H-5), 6.25 (t, 1 H, J=5.6 Hz, H-1′), 4.38(m, 1 H, H-3′), 3.95 (m, 2H, H-4′ and H-5′a), 3.78 (m, 1 H, H-5′), 2.50(m, 1 H, H-2′a), 2.10 (m, 1 H, H-2′b), 1.51 (s, 9 H, (CH₃)₃CO), 0.93 (s,9 H, (CH₃)₃CSi), 0.88 (s, 9 H, (CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si), 0.06(s, 6 H, (CH₃)₂Si).

N⁴-tert-Butyloxycarbonyl-N⁴-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.03)

NaH (32 mg, 1.26 mmol, dry) was added to a solution of compound dC.02(540 mg, 0.97 mmol) in anhydrous DMF (6 mL) at 0° C. and stirred for 30minutes under a nitrogen atmosphere. A solution of 2-nitrobenzyl bromide(313 mg, 1.45 mmol) in anhydrous DMF (1.5 mL) was added dropwise. Thereaction mixture was gradually warmed to room temperature and stirredovernight. Following the addition of ethyl acetate (60 mL), the mixturewas washed three times with saturated NH₄Cl solution (40 mL), and thecombined aqueous layer was extracted with ethyl acetate (40 mL). Thecombined organic layer was dried with Na₂SO₄, concentrated in vacuo, andpurified by silica gel chromatography to yieldN⁴-tert-butyloxycarbonyl-N⁴-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.03 (250 mg, 37%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.29 (d, 1 H, J=7.6 Hz, H-6), 8.05 (d, 1 H,J=8.0 Hz, Ph-H), 7.53 (t, 1 H, J=7.5 Hz, Ph-H), 7.38 (t, 1 H, J=7.6 Hz,Ph-H), 7.28 (m, 2 H, Ph-H and H-5), 6.26 (t, 1 H, J=5.6 Hz, H-1′), 5.60(q, 2 H, Ph-CH₂), 4.41 (m, 1 H, H-3′), 3.96 (m, 2 H, H-4′ and H-5′a),3.80 (m, 1 H, H-5′b), 2.51 (m, 1 H, H-2′a), 2.15 (m, 1H, H-2′b), 1.28(s, 9 H, (CH₃)₃CO), 0.95 (s, 9 H, (CH₃)₃CSi), 0.88 (s, 9 H, (CH₃)₃CSi),0.14 (s, 6 H, (CH₃)₂Si), 0.07 (s, 6 H, (CH₃)₂Si).

N⁴-(2-Nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.04)

Silica gel 60 (2.5 g, 100-200 mesh, activated by heating to 50-60° C.under reduced pressure for 24 hours) was added to a solution of compounddC.03 (250 mg, 0.36 mmol) in CH₂Cl₂ (5 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 60-70° C.under reduced pressure for 48 hours, washed three times with MeOH (30mL), and filtered using a buchi funnel. The combined filtrate wasconcentrated in vacuo and purified by silica gel chromatography to yieldN⁴-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.04 (0.185 g, 79%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.02 (d, 1 H, J=8.0 Hz, Ph-H), 7.92 (d, 1 H,J=7.2 Hz, H-6), 7.79 (d, 1 H, J=7.5 Hz, Ph-H), 7.57 (t, 1 H, J=7.3 Hz,Ph-H), 7.41 (t, 1 H, J=7.5 Hz, Ph-H), 6.38 (bs, 1 H, NH), 6.26 (t, 1 H,J=5.2 Hz, H-1′), 5.68 (d, 1 H, J=7.2 Hz, H-5), 4.92 (m, 2 H, Ph-CH₂),4.36 (m, 1 H, H-3′), 3.88 (m, 2 H, H-4′ and H-5′a), 3.75 (m, 1 H,H-5′b), 2.39 (m, 1 H, H-2′a), 2.07 (m, 1 H, H-2′b), 0.91 (s, 9 H,(CH₃)₃CSi), 0.87 (s, 9 H, (CH₃)₃CSi), 0.09 (s, 6 H, (CH₃)₂Si), 0.05 (s,6 H, (CH₃)₂Si).

N⁴-(2-Nitrobenzyl)-2′-deoxycytidine (dC.05)

A solution of n-NBu₄F (190 mg, 0.73 mmol) in THF (1.4 mL) was addeddropwise to a solution of compound dC.04 (170 mg, 0.29 mmol) in THF (3.4mL) at 0° C. under a nitrogen atmosphere. The reaction mixture wasstirred for two hours, concentrated in vacuo, and purified by silica gelchromatography to yield N⁴-(2-nitrobenzyl)-2′-deoxycytidine dC.05 (62mg, 59%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.23 (t, 1 H, D₂O exchangeable, NH), 8.07(d, 1 H, J=8.0 Hz, Ph-H), 7.83 (d, 1 H, J=7.4 Hz, H-6), 7.74 (t, 1 H,J=7.5 Hz, Ph-H), 7.54 (m, 2 H, Ph-H), 6.13 (t, 1 H, J=6.8 Hz, H-1′),5.91 (d, 1 H, J=7.4 Hz, H-5), 5.19 (d, 1 H, D₂O exchangeable, 3′-OH),4.97 (t, 1 H, D₂O exchangeable, 5′-OH), 4.78 (m, 2 H, Ph-CH₂), 4.19 (m,1 H, H-3′), 3.76 (m, 1 H, H-4′), 3.55 (m, 2 H, H-5′a and H-5′b), 2.09(m, 1 H, H-2′a), 1.93 (m, 1 H, H-2′b);

¹³C NMR (100 MHz, CD₃OD): δ 165.71, 158.51, 149.70, 141.75, 135.09,134.73, 131.30, 129.43, 125.94, 96.75, 88.83, 87.57, 72.03, 62.78,42.71, 42.00.

N⁴-(2-Nitrobenzyl)-2′-deoxycytidine-5′-triphosphate (WW2p044)

POCl₃ (17 μL, 0.2 mmol) was added to a solution of compound dC.05 (36mg, 0.1 mmol) and proton sponge (32 mg, 0.15 mmol) in trimethylphosphate(0.5 mL) at 0° C. and stirred for two hours. Additional POCl₃ (9 μL, 0.1mmol) was added and stirred for another hour. A solution ofbis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 μL) in anhydrous DMF (1 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×20 cm) with a lineargradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes) at a flow rate of4.5 mL/min. The fractions containing triphosphate were combined andlyophilized to give the triphosphate WW2p044 (34 mg, 52%) as a whitefluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.12 (d, 1 H, J=8.0 Hz, Ph-H), 7.83 (d, 1 H,J=7.6 Hz, H-6), 7.69 (t, 1 H, J=7.6 Hz, Ph-H), 7.60 (d, 1 H, J=7.6 Hz,Ph-H), 7.53 (t, 1 H, J=8.0 and 7.6 Hz, Ph-H), 6.29 (t, 1 H, J=6.8 Hz,H-1′), 6.15 (d, 1 H, J=7.6 Hz, H-5), 4.85 (bs, 2 H, Ph-CH₂), 4.58 (m, 1H, H-3′), 4.21 (m, 3 H, H-4′, H-5′a and H-5′b), 2.38 (m, 1 H, H-2′a),2.28 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.61 (d, J=15.9 Hz), −10.60 (d, J=15.4 Hz),−19.26 (br);

ToF-MS (ESI): For the molecular ion C₁₆H₂₁N₄O₁₅P₃Na [M+Na]⁺, thecalculated mass was 625.0114, and the observed mass was 624.9993.

Synthesis ofN4-[4-(3-amino-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidine-5′-triphosphateand dye labeling (WW2p080)

Scheme. Synthesis of 6-TAMRA labeledN⁴-[4-(3-amino-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidine-5′-triphosphate.(i) 4-iodo-2-nitrobenzyl bromide, n-Bu₄NBr, CH₂Cl₂, 1 M NaOH, roomtemperature, four hours, 45%; (ii) N-propargyltrifluoroacetamide,PdCl₂(PPh₃)₂, CuI, Et₃N, THF, reflux, three hours, 82%; (iii) SiO₂,vacuum, 70-80° C., 48 hours, 81%; (iv) n-Bu₄NF, THF, 0° C., two hours,then room temperature overnight, 39%; (v) POCl₃, proton sponge,(MeO)₃PO, 0° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes;1 M HNEt₃HCO₃, one hour; NH₄OH, one hour; 39%; (vi) 6-TAMRA-SE, 0.1 MNa₂CO₃/NaHCO₃ buffer (pH 9.2), one hour.

N⁴-tert-Butyloxycarbonyl-N⁴-(4-iodo-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.06)

A solution of 4-iodo-2-nitrobenzyl bromide (461 mg, 1.35 mmol) in CH₂Cl₂(4 mL) was added dropwise to a mixture of compound dC.02 (250 mg, 0.45mmol) and n-Bu₄NBr (145 mg, 0.45 mmol) in CH₂Cl₂ (4 mL) and NaOH (1 M;4.5 mL). The reaction was stirred vigorously at room temperature forfour hours. The organic layer was separated, and the aqueous layer wasextracted twice with CH₂Cl₂ (4 mL each). The combined organic layer wasdried with Na₂SO₄, concentrated in vacuo, and purified by silica gelchromatography to giveN⁴-tert-butyloxycarbonyl-N⁴-(4-iodo-2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.06 (167 mg, 45%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.37 (d, 1 H, J=1.8 Hz, Ph-H), 8.30 (d, 1 H,J=7.5 Hz, H-6), 7.82 (dd, 1 H, J=1.8 Hz and 8.3 Hz, Ph-H), 7.26 (d, 1 H,J=7.5 Hz, H-5), 6.99 (d, 1 H, J=8.4 Hz, Ph-H), 6.24 (dd, 1 H, J=6.3 and5.0 Hz, H-1′), 5.52 (q, 2 H, Ph-CH₂), 4.41 (m, 1 H, H-3′), 3.96 (m, 2 H,H-4′ and H-5′a), 3.80 (m, 1 H, H-5′b), 2.51 (m, 1 H, H-2′a), 2.14 (m, 1H, H-2′b), 1.34 (s, 9 H, (CH₃)₃CO), 0.95 (s, 9 H, (CH₃)₃CSi), 0.89 (s, 9H, (CH₃)₃CSi), 0.14 (s, 3 H, (CH₃)Si), 0.13 (s, 3 H, (CH₃)Si), 0.08 (s,3 H, (CH₃)Si), 0.07 (s, 3 H, (CH₃)Si).

N⁴-tert-Butyloxycarbonyl-N⁴-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.07)

Under a nitrogen atmosphere, a solution ofbis(triphenylphosphine)palladium(II) dichloride (41 mg, 0.058 mmol) inanhydrous THF (3 mL) was quickly added to a mixture of compound dC.06(315 mg, 0.39 mmol), CuI (15 mg, 0.078 mmol), Et₃N (0.73 mL, 5.2 mmol)and N-propargyltrifluoroacetamide (82 mg, 0.54 mmol) in anhydrous THF (7mL). The reaction mixture was refluxed for two hours, concentrated invacuo, and purified by silica gel chromatography to yieldN⁴-tert-butyloxycarbonyl-N⁴-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.07 (268 mg, 82%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.33 (d, 1 H, J=7.7 Hz, H-6), 8.03 (d, 1 H,J=1.5 Hz, Ph-H), 7.61 (bs, 1 H, NH), 7.50 (dd, 1 H, J=1.5 Hz and 8.2 Hz,Ph-H), 7.32 (d, 1 H, J=7.7 Hz, H-5), 7.18 (d, 1 H, J=8.2 Hz, Ph-H), 6.24(t, 1 H, J=6.0 Hz, H-1′), 5.56 (q, 2 H, PhCH₂), 4.42 (m, 1 H, H-3′),4.35 (d, 2 H, CH₂), 3.97 (m, 2 H, H-5′a and H-4′), 3.80 (m, 1 H, H-5′b),2.50 (m, 1 H, H-2′a), 2.05 (m, 1 H, H-2′b), 1.31 (s, 9 H, (CH₃)₃CO),0.96 (s, 9 H, (CH₃)₃CSi), 0.89 (s, 9 H, (CH₃)₃CSi), 0.15 (s, 3 H,(CH₃)Si), 0.14 (s, 3 H, (CH₃)Si), 0.07 (s, 6 H, (CH₃)₂Si).

N⁴-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidine(dC.08)

Silica gel 60 (3.1 g, 100-200 mesh, activated by heating to 50-60° C.under reduced pressure for 24 hours) was added to a solution of compounddC.07 (305 mg, 0.36 mmol) in CH₂Cl₂ (4 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 60-70° C.under reduced pressure for 24 hours, washed three times with MeOH (30mL), and filtered using a buchi funnel. The combined filtrate wasconcentrated in vacuo and purified by silica gel chromatography to yieldN⁴-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxycytidinedC.08 (219 mg, 81%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.25 (bs, 1 H, N—H), 7.94 (d, 1 H, J=7.4 Hz,H-6), 7.84 (s, 1 H, Ph-H), 7.62 (d, 1 H, J=7.7 Hz, Ph-H), 7.41 (d, 1 H,J=7.8 Hz, Ph-H), 6.25 (m, 2 H, N—H and H-1′), 5.67 (d, 1 H, J=7.3 Hz,H-5′), 4.83 (m, 2 H, Ph-CH₂), 4.38 (m, 2 H, CH₂ and H-3′), 3.90 (m, 2 H,H-4′ and H-5′a), 3.75 (m, 1 H, H-5′b), 2.36 (m, 1 H, H-2′a), 2.05 (m, 1H, H-2′b), 0.90 (s, 9 H, (CH₃)₃CSi), 0.87 (s, 9 H, (CH₃)₃CSi), 0.08 (s,6H, (CH₃)₂Si), 0.05 (s, 6 H, (CH₃)₂Si).

N⁴-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidine(dC.09)

A solution of n-Bu₄NF (94 mg, 0.36 mmol) in THF (2.5 mL) was addeddropwise to a solution of compound dC.08 (200 mg, 0.27 mmol) in THF (1mL) at 0° C. under a N₂ atmosphere. The reaction mixture was stirred at0° C. for two hours and then at room temperature overnight, concentratedin vacuo, and purified by silica gel chromatography to yieldN-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidinedC.09 (54 mg, 39%) as a white foam.

¹H NMR (400 MHz, (DMSO-d₆): δ 10.12 (t, 1 H, N—H), 8.26 (t, 1 H, N—H),8.08 (s, 1 H, Ph-H), 7.84 (d, 1 H, J=7.5 Hz, H-6), 7.78 (d, 1 H, J=8.1Hz, Ph-H), 7.50 (d, 1 H, J=8.1 Hz, Ph-H), 6.13 (t, 1 H, J=6.8 Hz, H-1′),5.91 (d, 1 H, J=7.4 Hz, H-5), 5.20 (d, 1 H, 3′-OH), 5.10 (t, 1 H,5′-OH), 4.77 (d, 2 H, Ph-CH₂), 4.35 (m, 1 H, H-3′), 4.31 (m, 2 H, CH₂),3.80 (m, 1 H, H-4′), 3.55 (m, 2 H, H-5′), 2.10 (m, 1 H, H-2′a), 1.92 (m,1 H, H-2′b).

N⁴-[4-(3-Amino-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidine-5′-triphosphate(dC.10)

POCl₃ (18 μL, 0.2 mmol) was added to a solution of compound dC.09 (49mg, 0.1 mmol) and proton sponge (32 mg, 0.15 mmol) in trimethylphosphate(0.5 mL) at 0° C. and stirred for two hours. A solution ofbis-tri-n-butylammonium pyrophosphate (237 mg, 0.5 mmol) andtri-n-butylamine (100 uL) in anhydrous DMF (1 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature, followed by the dropwise addition of concentrated ammoniumhydroxide (5 mL, 27%) at 0° C. The mixture was stirred for an additionalhour at room temperature and then lyophilized to dryness. The residuewas dissolved in water (10 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×20 cm) with a lineargradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes) at a flow rate of4.5 mL/min. The fractions containing triphosphate were combined andlyophilized to give the triphosphate dC.10 (28 mg, 39%) as a whitefluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.22 (s, 1 H, Ph-H), 7.88 (d, 1 H, J=7.6 Hz,H-6), 7.74 (d, 1 H, J=8.0 Hz, Ph-H), 7.59 (d, 1 H, J=8.0 Hz, Ph-H), 6.33(t, 1 H, J=6.8 Hz, H-1′), 6.18 (d, 1 H, J=7.6 Hz, H-5), 4.61 (m, 1 H,H-3′), 4.24 (m, 3 H, H-4′, H-5′), 3.63 (s, 2 H, CH₂), 2.42 (m, 1 H,H-2′a), 2.29 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ-5.88 (d, J=15.6 Hz), −10.69 (d, J=15.6 Hz),−19.25 (t, J=15.6 Hz);

ToF-MS (ESI): For the molecular ion C₁₉H₂₂N₅O₁₅P₃Na [M-2H+Na]⁻, thecalculated mass was 676.0223, and the observed mass was 676.0563.

6-TAMRA LabeledN⁴-[4-(3-Amino-1-propynyl)-2-nitrobenzyl]-2′-deoxycytidine-5′-triphosphate(WW2p080)

A solution of 6-TAMRA-SE (0.75 mg, 1.4 μmol) in anhydrous DMSO (30 μL)was added to a solution of triphosphate dC.10 (1.6 μmol) inNa₂CO₃/NaHCO₃ buffer (0.1 M, pH 9.2; 0.3 mL) and incubated at roomtemperature for 30 minutes. The reaction was purified with reverse-phaseHPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm) to yield the6-TAMRA labeled triphosphate WW2p080. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved a linear gradient of5-50% B for 40 minutes and then 50-90% B for 10 minutes. Theconcentration of WW2p080 was estimated by adsorption spectroscopy usingthe extinction coefficient of the 6-TAMRA dye (i.e., 65,000 at 555 nm).

Synthesis ofN4-[1-(2-nitrophenyl)ethyl]-2′-deoxycytidine-5′-triphosphate (WW2p115)

Scheme. Synthesis ofN⁴-[1-(2-nitrophenyl)ethyl]-2′-deoxycytidine-5′-triphosphate. (i) TBSCl,imidiazole, DMF, room temperature, three hours, 92%; (ii) TPSCl, DMAP,CH₂Cl₂, room temperature, 88%; (iii) 1-(2-nitrophenyl)ethylamine dC.13c,DMF, 90° C., 1.5 hours, 39%; (iv) n-Bu₄NF, THF, 0° C., two hours, thenroom temperature, one hour, 90%; (v) POCl₃, proton sponge, (MeO)₃PO, 0°C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 MHNEt₃HCO₃, one hour.

3′,5′-O-Bis-tert-butyldimethylsilyl-2′-deoxyuridine (dC.11)

Under a nitrogen atmosphere, a mixture of 2′-deoxyuridine dU (2.5 g,10.95 mmol), TBSCl (7.26 g, 48.2 mmol) and imidiazole (6.56 g, 96.4mmol) in anhydrous DMF (25 mL) was stirred at 0° C. for two hours andthen warmed to room temperature over one hour. The reaction mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield 3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyuridine dC.11 (4.62g, 92%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 9.48 (s, 1 H, NH), 7.80 (d, 1 H, J=8.1 Hz,H-6), 6.19 (t, 1 H, J=6.4 Hz, H-1′), 5.59 (d, 1 H, J=8.1 Hz, H-5), 4.31(m, 1 H, H-3′), 3.81 (m, 2 H, H-4′ and H-5′a), 3.65 (m, 1 H, H-5′b),2.21 (m, 1 H, H-2′a), 1.97 (m, 1 H, H-2′b), 0.81 (s, 9 H, (CH₃)₃CSi),0.79 (s, 9 H, (CH₃)₃CSi), 0.00 (s, 6 H, (CH₃)₂Si), −0.02 (2s, 3 H each,(CH₃)₂Si).

O⁴-(2,4,6-Triisopropylbenzenesulfonyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyuridine(dC.12)

2,4,6-Triisopropylbenzenesulfonyl chloride (660 mg, 2.19 mmol) was addedto a solution of compound dC.11 (500 mg, 1.09 mmol), DMAP (6.7 mg,catalytic amount) and Et₃N (0.62 mL, 4.38 mmol) in anhydrous CH₂Cl₂ (6mL) under a nitrogen atmosphere. The reaction mixture was stirredovernight at room temperature, concentrated in vacuo, and purified bysilica gel column chromatography to yieldO⁴-(2,4,6-triisopropylbenzenesulfonyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyuridinedC.12 (690 mg, 88%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.46 (d, 1 H, J=7.3 Hz, H-6), 7.20 (s, 2 H,Ph-H), 6.08 (dd, 1 H, J=4.3 Hz, H-1′), 6.01 (d, 1 H, J=7.3 Hz, H-5),4.33 (m, 1 H, H-3′), 4.25 (m, 2 H, CH), 3.94 (m, 2 H, H-4′ and H-5′a),3.76 (m, 1 H, H-5′b), 2.91 (m, 1 H, CH), 2.48 (m, 1 H, H-2′a), 2.12 (m,1 H, H-2′b), 1.31 (d, 6 H, CH₃), 1.26 (dd, 12 H, CH₃), 0.91 (s, 9 H,(CH₃)₃CSi), 0.86 (s, 9 H, (CH₃)₃CSi), 0.10 and 0.09 (2 s, 6 H,(CH₃)₂Si), 0.04 (s, 6 H, (CH₃)₂Si).

N⁴-[1-(2-nitrophenyl)ethyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′deoxycytidine(dC.13)

A solution of compound dC.12 (410 mg, 0.56 mmol) and1-(2-nitrophenyl)ethylamine dC.13c (470 mg, 2.82 mmol) in anhydrous DMF(3 mL) was heated to 90° C. for 1.5 hours and then concentrated invacuo. The residue was dissolved in CH₂Cl₂ (50 mL), washed withsaturated NH₄Cl solution (30 mL), with water (30 mL), and with saturatedNaHCO₃ solution (30 mL). The organic layer was dried with Na₂SO₄,filtered, concentrated in vacuo, and purified by silica gel columnchromatography to yieldN⁴-[1-(2-nitrophenyl)ethyl]-3′,5′-O-bis-tert-butyldimethylsilyl-2′deoxycytidine dC.13 (130 mg, 39%, 1:1 mixture of diastereomers) as awhite foam.

¹H NMR (400 MHz, CDCl₃) for diastereomers: δ 7.97 (m, 1 H, Ph-H), 7.89(d, 1 H, J=7.1 Hz, H-6), 7.65 (m, 2 H, Ph-H), 7.41 (m, 1 H, Ph-H), 6.19(m, 1 H, H-1′), 5.91 (m, 1 H, H-5), 5.37 (m, 1 H, Ph-CH), 4.34 (m, 1 H,H-3′), 3.86 (m, 2 H, H-4′ and H-5′a), 3.72 (m, 1 H, H-5′b), 2.05 (m, 1H, H-2′a), 1.83 (m, 1 H, H-2′b), 1.59 (bs, 3 H, CH₃), 0.86 (s, 12 H,(CH₃)₃CSi), 0.03 (s, 12 H, (CH₃)₂Si).

N⁴-[1-(2-nitrophenyl)ethyl]-2′-deoxycytidine (dC.14)

A solution of n-Bu₄NF (142 mg, 0.54 mmol) in THF (3 mL) was added to asolution of compound dC.13 (130 mg, 0.22 mmol) in THF (2 mL) at 0° C.The reaction mixture was stirred at 0° C. for two hours and then at roomtemperature for one hour, concentrated in vacuo, and purified by silicagel column chromatography to yieldN⁴-[1-(2-nitrophenyl)ethyl]-2′-deoxycytidine dC.14 (80 mg, 90%, 1:1mixture of diastereomers) as a white powder.

¹H NMR (400 MHz, CD₃OD) for diastereomers: δ 7.95 (m, 2 H, Ph-H andH-5), 7.65 (m, 2 H, Ph-H), 7.45 (m, 1 H, Ph-H), 6.22 (m, 1 H, H-1′),5.97 (m, 1 H, H-5), 5.74 (m, 1 H, Ph-CH), 4.34 (m, 1 H, H-3′), 3.93 (m,1 H, H-4′), 3.75 (m, 2 H, H-5′), 2.32 (m, 1 H, H-2′a), 2.09 (m, 1 H,H-2′b), 1.59 (m, 3 H, CH₃).

N⁴-[1-(2-nitrophenyl)ethyl]-2′-deoxycytidine-5′-triphosphate (WW2p115)

POCl₃ (14 μL, 0.15 mmol) was added to a solution of compound dC.14 (28mg, 0.08 mmol) and proton sponge (23 mg, 0.11 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (180 mg, 0.38 mmol)and tri-n-butylamine (80 μL) in anhydrous DMF (0.8 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×20 cm) with a lineargradient of NH₄HCO₃ (50 mM to 500 mM in 300 minutes) at a flow rate of4.5 mL/min. The fractions containing triphosphate were combined andlyophilized to give the triphosphate WW2p115 (24 mg, 47%, 1:1 mixture ofdiastereomers) as a white fluffy solid.

¹H NMR (400 MHz, D₂O) for diastereomers: δ 7.98 (m, 1 H, Ph-H), 7.80 (m,1 H, H-6), 7.66 (m, 2 H, Ph-H), 7.49 (m, 1 H, Ph-H), 6.24 (m, 1 H,H-1′), 6.11 (m, 1 H, H-5), 5.60 (m,1 H, Ph-CH), 4.55 (m, 1 H, H-3′),4.19 (m, 3 H, H-4′and H-5′), 2.35 (m, 1 H, H-2′a), 2.24 (m, 1 H, H-2′b),1.59 (d, 3 H, J=6.7 Hz, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: 6-6.00 (d, J=14.1 Hz), −10.82(d, J=15.6 Hz), −19.36 (t, J=15.9 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₃N₄O₁₅P₃Na [M+Na]⁺, thecalculated mass was 639.0271, and the observed mass was 639.0332.

Synthesis of 1-(2-nitrophenyl)ethylamine (dC.13c)

Scheme for dC.13c. Synthesis of 1-(2-nitrophenyl)ethylamine. (i) NaBH₄,MeOH, dioxane, room temperature, one hour, 92%; (ii) Phthalimide, DIAD,Ph₃P, THF, 0° C., three hours, 99%; (iii) NH₂NH₂, CH₃CH₂OH, reflux, onehour, 80%.

1-(2-Nitrophenyl)ethanol (dC.13a)

NaBH₄ (3.24 g, 85.60 mmol) was slowly added to a solution of2′-nitroacetophenone NAP (3.74 g, 22.65 mmol) in a mixture of methanol(34 mL) and dioxane (22 mL). The reaction mixture was stirred at roomtemperature for one hour and then diluted with ethyl acetate (100 mL)and washed with water (25 mL). The organic layer was dried with Na₂SO₄,filtered, and concentrated in vacuo to yield 1-(2-nitrophenyl)ethanoldC.13a (3.49 g, 92%) as a white powder.

¹H NMR (400 MHz, CDCl₃): δ 7.89 (d, 1 H, J=8.1 Hz, Ph-H), 7.83 (d, 1 H,J=7.4 Hz, Ph-H), 7.65 (t, 1 H, J=7.4 Hz, Ph-H), 7.42 (t, 1 H, J=8.1 Hz,Ph-H), 5.41 (q, 1 H, J=6.0 Hz, Ph-CH), 2.48 (s, 1 H, OH), 1.57 (d, 3 H,J=6.4 Hz, CH₃).

N-[1-(2-nitrophenyl)ethyl]phthalimide (dC.13b)

Phthalimide (660 mg, 4.5 mmol) was added to a solution of compounddC.13a (750 mg, 4.5 mmol) and Ph₃P (1.41 g, 5.4 mmol) in THF (12 mL).The suspension was cooled to 0° C. and stirred for 10 minutes, and thendiisopropyl azodicarboxylate (1.1 mL, 5.4 mmol) was added dropwise.After stirring at 0° C. for three hours, the reaction mixture wasconcentrated in vacuo and purified by silica gel column chromatographyto yield N-[1-(2-nitrophenyl)ethyl]phthalimide dC.13b (1.33 g, 99%) as abrown oil.

¹H NMR (400 MHz, CDCl₃): δ 7.93 (d, 1 H. J=7.9 Hz, Ph-H), 7.81 (m, 3 H,Ph-H), 7.71 (m, 2 H, Ph-H), 7.61 (t, 1 H, J=7.6 Hz, Ph-H), 7.44 (t, 1 H,J=7.6 Hz, Ph-H), 6.08 (q, 1 H, J=7.2 Hz, Ph-CH), 1.97 (d, 3 H, J=7.2 Hz,CH₃).

1-(2-nitrophenyl)ethylamine (dC.13c)

Compound dC.13b (1.33 g, 4.5 mmol) was dissolved in ethanol (21 mL) uponheating to 50° C. and then cooled to room temperature. Hydrazine (0.55mL, 11.22 mmol) was added, and the reaction mixture was refluxed for onehour and then cooled with ice. Diethyl ether (40 mL) was added toprecipitate the compound, which was isolated by filtration and washedtwo times with diethyl ether (40 mL each). The combined filtrate waswashed two times with water (40 mL each) and then with brine (40 mL).The organic layer was dried over Na₂SO₄ and concentrated in vacuo toyield 1-(4-iodo-2-nitrophenyl)ethylamine dC.13c (600 mg, 80%) as a brownoil.

¹H NMR (400 MHz, CDCl₃): δ 7.78 (m, 2 H. Ph-H), 7.61 (t, 1 H, J=7.6 Hz,Ph-H), 7.37 (t, 1 H, J=7.6 Hz, Ph-H), 4.59 (q, 1 H, J=6.4 Hz, Ph-CH),1.45 (d, 3 H, J=6.4 Hz, CH₃).

Synthesis of N4-[1-(2-nitrophenyl)ethyl]-cytidine-5′-triphosphate(WW2p152 and WW3p026)

Scheme. Synthesis ofN⁴-[1-(2-nitrophenyl)ethyl]-cytidine-5′-triphosphate. (i) TBSCl,imidiazole, DMF, room temperature, 60 hours, 95%; (ii) TPSCl, Et₃N,DMAP, CH₂Cl₂, room temperature, overnight, 80%; (iii)1-(2-nitrophenyl)ethylamine, DMF, 90° C., 45 minutes; n-Bu₄NF, THF, 0°C., then gradually warmed to room temperature, two hours; 60% for twodiastereoisomers; (iv) POCl₃, proton sponge, (MeO)₃PO, 0° C., two hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, one hour.

2′,3′,5′-O-Tris-tert-butyldimethylsilyl-uridine (C.1)

TBSCl (995 mg, 6.6 mmol) was added to a solution of uridine (244 mg, 1mmol) and imidiazole (898 mg, 13.2 mmol) in anhydrous DMF (5 mL) at 0°C. under a nitrogen atmosphere. The mixture was warmed to roomtemperature and stirred for 60 hours. Ethyl acetate (30 mL) was added,and the mixture was washed two times with saturated NH₄Cl solution (20mL each) and with water (20 mL), dried over Na₂SO₄, concentrated andpurified by silica gel column chromatography to yield2′,3′,5′-O-tris-tert-butyldimethylsilyl-uridine C.1 (558 mg, 95%) as awhite foam.

¹H NMR (400 MHz, CDCl₃): δ 8.79 (s, 1 H, NH), 8.03 (d, 1 H, J=8.2 Hz,H-6), 5.87 (d, 1 H, J=3.5 Hz, H-1′), 5.68 (d, 1 H, J=8.2 Hz, H-5), 4.08(m, 3 H, H-2′, H-3′ and H-4′), 3.99 (d, 1 H, J=11.6 Hz, H-5′a), 3.77 (d,1 H, J=11.6 Hz, H-5′b), 0.95 (s, 9 H, (CH₃)₃CSi), 0.91 and 0.90 (2 s, 18H, (CH₃)₃CSi), 0.09 (5 s, 18 H, (CH₃)₂Si).

O⁴-(2,4,6-Triisopropylbenzenesulfonyl)-2′,3′,5′-O-tris-tert-butyldimethylsilyl-uridine(C.2)

2,4,6-Triisopropylbenzenesulfonyl chloride (557 mg, 1.84 mmol) was addedto a solution of compound C.1 (540 mg, 0.92 mmol), DMAP (12 mg,catalytic amount) and Et₃N (0.5 mL, 3.68 mmol) in anhydrous CH₂Cl₂ (10mL) under a nitrogen atmosphere. The mixture was stirred overnight atroom temperature, CH₂Cl₂ (20 mL) was added, washed with saturated NH₄Clsolution (15 mL), dried over Na₂SO₄, concentrated and purified by silicagel column chromatography to yieldO⁴-(2,4,6-triisopropyl-benzenesulfonyl)-2′,3′,5′-O-tris-tert-butyldimethylsilyl-uridineC.2 (629 mg, 80%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.62 (d, 1 H, J=7.3 Hz, H-6), 7.20 (s, 2 H,Ph-H), 5.99 (d, 1 H, J=7.3 Hz, H-5), 5.68 (d, 1 H, J=1.0 Hz, H-1′), 4.23(m, 2 H, CH), 4.09 (m, 3 H, H-2′, H-3′ and H-4′), 4.00 (m, 1 H, H-5′a),3.78 (1 H, d, J=11.8 Hz, H-5′b), 2.90 (m, 1 H, CH), 1.31 (d, 6 H, CH₃),1.26 (dd, 12 H, CH₃), 0.94 (s, 9 H, (CH₃)₃CSi), 0.88 (2 s, 18 H,(CH₃)₃CSi), 0.17-0.05 (6 s, 18 H, (CH₃)₂Si).

N⁴-[1-(2-Nitrophenyl)ethyl]-cytidine (Single Diastereoisomer C.3 ds1 andC.3 ds2)

A solution of compound C.2 (476 mg, 0.56 mmol) and1-(2-nitrophenyl)ethylamine dC.13c (498 mg, 3 mmol) in anhydrous DMF (4mL) was heated at 90° C. for 45 minutes. Ethyl acetate (40 mL) wasadded, the mixture was washed with saturated NH₄Cl solution (20 mL) andwater (20 mL), and dried with Na₂SO₄, concentrated. The twodiastereoisomers were separated by silica gel column chromatography toyieldN⁴-[1-(2-nitrophenyl)ethyl]-2′,3′,5′-O-bis-tert-butyldimethylsilyl-cytidinesingle diastereo-isomer ds1 (fast eluting, 290 mg) and ds2 (sloweluting, 203 mg). Both single diastereoisomers were used in the nextstep without further purification.

N⁴-[(R or S)-1-(2-nitrophenyl)ethyl]-cytidine (Single DiastereoisomerC.3 ds1)

A solution of n-Bu₄NF (235 mg, 0.9 mmol) in THF (3 mL) was added to asolution ofN⁴-[1-(2-nitrophenyl)ethyl]-2′,3′,5′-O-bis-tert-butyldimethylsilyl-cytidinesingle diastereo-isomer ds1 (264 mg) in THF (4 mL) at 0° C. The reactionmixture was gradually warmed to room temperature and stirred for twohours. Silica gel 60 (1 g) was added, and the mixture was evaporated invacuo to dryness. The residue was purified by silica gel columnchromatography to yield N⁴-[(R or S)-1-(2-nitrophenyl)ethyl]-cytidinesingle diastereoisomer C.3 ds1 (65 mg, ca. 30% for two steps, absoluteconfiguration not determined) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.39 (d, 1 H, D₂O exchangeable, NH), 7.92(dd, 1 H, J=1.1 and 8.1 Hz, Ph-H), 7.82 (d, 1 H, J=7.5 Hz, H-6), 7.70(dt, 1 H, J=1.1 and 7.5 Hz, Ph-H), 7.64 (dd, 1 H, J=1.2 and 7.5 Hz,Ph-H), 7.49 (dt, 1 H, J=1.3 and 7.5 Hz, Ph-H), 1 H, Ph-H), 5.80 (d, 1 H,J=7.5 Hz, H-5), 5.65 (d, 1 H, J=3.4 Hz, H-1′), 5.50 (m, 1 H, Ph-CH),5.30 (d, 1 H, D₂O exchangeable, 3′-OH), 5.02 (t, 1 H, D₂O exchangeable,5′-OH), 4.96 (d, 1 H, D₂O exchangeable, 2′-OH), 3.90 (m, 2 H, H-2′ andH-3′), 3.78 (m, 1 H, H-4′), 3.59 (m, 1 H, H-5′a), 3.50 (m, 1 H, H-5′b),1.48 (d, 3 H, J=6.9 Hz, CH₃).

N⁴-[(S or R)-1-(2-nitrophenyl)ethyl]-cytidine (Single DiastereoisomerC.3 ds2)

A solution of n-Bu₄NF (167 mg, 0.64 mmol) in THF (2 mL) was added to asolution ofN⁴-[1-(2-nitrophenyl)ethyl]-2′,3′,5′-O-bis-tert-butyldimethylsilyl-cytidinesingle diastereo-isomer ds2 (188 mg) in THF (3 mL) at 0° C. The reactionmixture was gradually warmed to room temperature and stirred for onehour. Silica gel 60 (1 g) was added, and the mixture was evaporated invacuo to dryness. The residue was purified by silica gel columnchromatography to yield N⁴-[(S or R)-1-(2-nitrophenyl)ethyl]-cytidinesingle diastereoisomer C.3 ds2 (67 mg, ca. 30% for two steps, absoluteconfiguration not determined) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 8.38 (d, 1 H, D₂O exchangeable, NH), 7.92(dd, 1 H, J=1.1 and 8.1 Hz, Ph-H), 7.80 (d, 1 H, J=7.5 Hz, H-6), 7.72(dt, 1 H, J=1.0 and 7.5 Hz, Ph-H), 7.63 (dd, 1 H, J=1.1 and 7.5 Hz,Ph-H), 7.49 (dt, 1 H, J=1.3 and 7.5 Hz, Ph-H), 5.79 (d, 1 H, J=7.5 Hz,H-5), 5.68 (d, 1 H, J=4.0 Hz, H-1′), 5.51 (m, 1 H, Ph-CH), 5.20 (d, 1 H,D₂O exchangeable, 3′-OH), 5.01 (t, 1 H, D₂O exchangeable, 5′-OH), 4.93(d, 1 H, D₂O exchangeable, 2′-OH), 3.87 (m, 2 H, H-2′ and H-3′), 3.78(m, 1 H, H-4′), 3.59 (m, 1 H, H-5′a), 3.50 (m, 1 H, H-5′a), 1.49 (d, 3H, J=6.9 Hz, CH₃).

N⁴-[(R or S)-1-(2-Nitrophenyl)ethyl]-cytidine-5′-triphosphate SingleDiastereoisomer (WW3p026)

POCl₃ (14 μL, 0.15 mmol) was added to a solution of compound C.3 ds1 (29mg, 0.074 mmol) and proton sponge (32 mg, 0.15 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (175 mg, 0.37 mmol)and tri-n-butylamine (74 μL) in anhydrous DMF (0.74 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), and part of the solution was purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yield N⁴-[(R or S)-1-(2-nitrophenyl)ethyl]-cytidine-5′-triphosphatesingle diastereoisomer WW3p026 (absolute configuration not determined).Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved a linear gradient of 5-50% B for 20 minutes and then 50-90% Bfor 10 minutes.

¹H NMR (400 MHz, D₂O): δ 8.0 (d, 1 H, J=8.1 Hz, Ph-H), 7.92 (d, 1 H,J=7.6 Hz, H-6), 7.70 (m, 2 H, Ph-H), 7.50 (t, 1 H, J=8.0 Hz, Ph-H), 6.14(d, 1 H, J=7.6 Hz, H-5), 5.92 (d, 1 H, J=4.1 Hz, H-1′), 5.61 (q, 1 H,J=6.8 Hz, Ph-CH), 4.33-4.21 (m, 3 H, H-2′, H-3′ and H-4′), 4.01 (m, 2 H,H-5′), 1.60 (d, 3 H, J=6.8 Hz, CH₃);

N⁴-[(S or R)-1-(2-nitrophenyl)ethyl]-cytidine-5′-triphosphate SingleDiastereoisomer (WW2p152)

POCl₃ (11 μL, 0.12 mmol) was added to a solution of compound C.3 ds2 (31mg, 0.08 mmol) and proton sponge (26 mg, 0.12 mmol) intrimethylphosphate (0.5 mL) at 0° C. and stirred for two hours. Asolution of bis-tri-n-butylammonium pyrophosphate (190 mg, 0.4 mmol) andtri-n-butylamine (80 μL) in anhydrous DMF (0.8 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and purified by anion exchangechromatography using a Q Sepharose FF column (2.5×20 cm) with a lineargradient of NH₄HCO₃ (50 mM to 500 mM in 240 minutes) at a flow rate of4.5 mL/min. The fractions containing triphosphate were combined andlyophilized to yield N⁴-[(S orR)-1-(2-nitrophenyl)ethyl]-cytidine-5′-triphosphate singlediastereoisomer WW2p152 (26 mg, 47%, absolute configuration notdetermined) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 7.99 (d, 1 H, J=8.2 Hz, Ph-H), 7.82 (d, 1 H,J=7.6 Hz, H-6), 7.68 (m, 2 H, Ph-H), 7.50 (t, 1 H, J=7.8 Hz, Ph-H), 6.12(d, 1 H, J=7.5 Hz, H-5), 5.91 (d, 1 H, J=4.4 Hz, H-1′), 5.60 (q,1 H,J=6.8 Hz, Ph-CH), 4.26 (m, 5 H, H-2′, H-3′, H-4′ and H-5′), 1.60 (d, 3H, J=6.8 Hz, CH₃);

³¹P NMR (162 MHz, D₂O): 6-5.18(d, J=19.8 Hz), −10.46 (d, J=19.1 Hz),−20.98 (t, J=19.6 Hz);

ToF-MS (ESI): For the molecular ion C₁₇H₂₁N₄O₁₆P₃Na [M-2H+Na]⁻, thecalculated mass was 653.0063, and the observed mass was 652.9975.

Synthesis of5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxycytidine-5′-triphosphate(WW3p###)

Scheme. Synthesis of5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxycytidine-5′-triphosphate.(i) TBSCl, imidazole, DMF, room temperature, 24 hours, 42%; (ii) TPSCl,DMAP, Et₃N, CH₂Cl₂, room temperature, 48 hours, 56%; (iii) NH₃,1,4-dioxane, 80° C., two hours, 56%; (iv) n-Bu₄NF, THF, roomtemperature; (v) POCl₃, proton sponge, (MeO)₃PO, 0° C.;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, one hour.

3′,5′-Bis-tert-butyldimethylsilyl-5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxyurdine(dC.15)

A solution of TBSCl (114 mg, 0.76 mmol) in anhydrous DMF (0.5 mL) wasadded to a solution of compound dT.07 (97 mg, 0.24 mmol) and imidiazole(103 mg, 1.52 mmol) in anhydrous DMF (1 mL). The mixture was stirred atroom temperature for 24 hours under a nitrogen atmosphere, thenconcentrated in vacuo and purified by silica gel column chromatographyto yield3′,5′-bis-tert-butyldimethylsilyl-5-[1-(2-nitrophenyl)-ethyloxymethyl]-2′-deoxyurdinedC.15 (64 mg, 42%, 1:1 mixture of diastereomers).

¹H NMR (400 MHz, CDCl₃) for diastereomers: δ 9.39 and 9.33 (2 br s, 1 H,NH), 7.92 (m, 2 H, Ph-H), 7.70 (m, 2 H, H-6 and Ph-H), 7.42 (m, 1 H,Ph-H), 6.29 (m, 1 H, H-1′), 5.11 (m, 1 H, Ph-CH), 4.41 (m, 1 H, H-3′),4.04 (m, 2 H, CH₂), 3.96 (m, 1 H, H-4′), 3.80 (m, 2 H, H-5′), 2.38 (m, 1H, H-2′a), 2.04 (m, 1 H, H-2′b), 1.55 (m, 3 H, CH₃), 0.91 (s, 18 H,(CH₃)₃CSi), 0.10 (s, 12 H, (CH₃)₂Si);

¹³C NMR (100 MHz, CDCl₃) for diastereomers: δ 162.89/162.76 (C), 150.15(C), 148.40 (C), 139.21/139.17 (C), 138.94/138.68 (CH), 133.86/133.76(CH), 128.22/128.19 (CH), 128.17/128.07 (CH), 124.23 (CH), 111.30/111.22(C), 87.97/87.94 (CH), 85.43/85.37 (CH), 73.46/73.43 (CH), 72.34/72.28(CH), 63.92/63.77 (CH₂), 63.07 (CH₂), 41.27/41.17 (CH₂), 25.97/25.93(CH₃), 23.69/23.57 (CH₃), 18.43/18.41 (C), −4.64/−4.82 (CH₃),−5.37/−5.40 (CH₃).

ES+ MS (ESI): 658 [M+Na]⁺;

O⁴-(2,4,6-Triisopropylbenzenesulfonyl)-3′,5′-bis-tert-butyldimethylsilyl-5-[1-(2-nitro-phenyl)ethyloxymethyl]-2′-deoxyurdine(dC.16)

A solution of 2,4,6-triisopropylbenzenesulfonyl chloride (61 mg, 0.20mmol) was added to a solution of dC.15 (64 mg, 0.10 mmol) and DMAP (6mg, 0.05 mmol) in anhydrous CH₂Cl₂ (3 mL) followed by Et₃N (63 μL, 0.45mmol). The mixture was stirred at room temperature for 48 hours under anitrogen atmosphere, then concentrated in vacuo and purified by silicagel column chromatography to giveO⁴-(2,4,6-triisopropylbenzenesulfonyl)-3′,5′-bis-tert-butyldimethylsilyl-5-[1-(2-nitrophenyl)ethyloxy-methyl]-2′-deoxyurdinedC.16 (50 mg, 56%, 1:1 mixture of diastereomers).

¹H NMR (400 MHz, CDCl₃) for diastereomers: δ 8.33 and 8.28 (2 s, 1 H,Ph-H), 7.90 (m, 3 H, Ph-H), 7.67 (m, 2 H, H-6 and Ph-H), 7.44 (m, 1 H,Ph-H), 6.27 (m, 1 H, H-1′), 5.11 (m, 1 H, Ph-CH), 4.40 (m, 1 H, H-3′),4.06 (m, 2 H, CH₂), 3.97 (m, 1 H, H-4′), 3.79 (m, 2 H, H-5′), 2.38 (m, 1H, H-2′a), 2.04 (m, 1 H, H-2′b), 1.54 (m, 3 H, CH₃), 1.60 (m, 3 H, CH),0.91 (s, 18 H, (CH₃)₃CSi), 0.80 (m, 18 H, CH₃), 0.09 (s, 12 H,(CH₃)₂Si);

3′,5′-Bis-tert-butyldimethylsilyl-5-[1-(2-nitrophenyl)ethyloxymethyl]-2′-deoxycytidine(dC.17)

A solution of NH₃ (1 mL, 0.5 M in dioxane) was added to a solution ofcompound dC.16 (48 mg, 0.05 mmol) in anhydrous 1,4-dioxane (1 mL). Themixture was stirred at 80° C. for two hours, then concentrated in vacuoand purified by silica gel column chromatography to give3′,5′-bis-tert-butyldimethylsilyl-5-[1-(2-nitrophenyl)ethyloxy-methyl]-2′-deoxycytidinedC.18 (25 mg, 58%, 1:1 mixture of diastereomers).

Example 4 dG Compounds Synthesis ofN²-(2-nitrobenzyl)-2′-deoxyguanidine-5′-triphosphate (WW2p067)

Scheme. Synthesis ofN²-(2-nitrobenzyl)-2′-deoxyguanidine-5′-triphosphate. (i) TBSCl,imidazole, anhydrous DMF, 0° C., then gradually warmed to roomtemperature, overnight, 96%; (ii) Ac₂O, anhydrous pyridine, roomtemperature, 100° C., 72%; (iii) Boc₂O, Et₃N, DMAP, CH₂Cl₂, reflux, twohours, 54%; (iv) K₂CO₃, MeOH, 95%; (v). 2-nitrobenzylbromide, NaH, DMF,0° C., then gradually warmed to room temperature, one hour, 91%; (vi)SiO₂, vacuum, 70-80° C., 48 hours, 97%; (vii) n-Bu₄NF, THF, 0° C., onehour, then room temperature, two hours, 83%; (viii) POCl₃, (MeO)₃PO,minus 20-30° C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes;1 M HNEt₃HCO₃, one hour, 62%.

3′,5′-O-Bis-tert-butyldimethylsilyl-2′-deoxyguanosine (dG.01)

2′-deoxyguanosine dG (0.89 g, 3.30 mmol), imidiazole (2.0 g, 29.32mmol), and TBSCl (2.34 g, 15.55 mmol) were added to anhydrous DMF (8 mL)at 0° C. under a N₂ atmosphere. The reaction mixture was graduallywarmed to room temperature and stirred overnight. The mixture was thenconcentrated in vacuo, treated with a mixture of CHCl₃ (8 mL) and MeOH(8 mL), concentrated in vacuo, and purified by silica gel chromatographyto yield 3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanosine dG.01(1.57 g, 96%) as a white powder.

¹H NMR (400 MHz, DMSO-d₆): δ 10.60 (br s, 1 H, H-1), 7.88 (s, 1 H, H-8),6.47 (br s, 2 H, NH₂), 6.10 (t, 1 H, J=6.8 Hz, H-1′), 4.48 (m, 1 H,H-3′), 3.80 (m, 1 H, H-4′), 3.70 (m, 2 H, H-5′a and H-5′b), 2.64 (m, 1H, H-2′a), 2.18 (m, 1 H, H-2′b), 0.89 (s, 9 H, (CH₃)₃CSi), 0.86 (s, 9 H,(CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si), 0.03 (s, 6 H, (CH₃)₂Si).

N²-Acetyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanosine (dG.02)

Acetic anhydride (1.22 mL, 12.92 mmol) was slowly added to a solution ofcompound dG.01 (0.51 g, 1.03 mmol) in anhydrous pyridine (10 mL) at roomtemperature and stirred at 100° C. for three hour. The reaction wasconcentrated in vacuo, co-evaporated three times with anhydrous ethanol(15 mL), and purified by silica gel chromatography to yieldN²-acetyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanosine dG.02(0.34 g, 56%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 12.03 (s, 1 H, NH), 11.70 (s. 1 H, NH),8.20 (s, 1 H, H-8), 6.19 (t, 1 H, J=6.2 Hz, H-1′), 4.51 (m, 1 H, H-3′),3.84 (m, 1 H, H-4′), 3.68 (m, 2 H, H-5′a and H-5′b), 2.71 (m, 1 H,H-2′a), 2.17 (m, 1 H, H-2′b), 0.89 (s, 9 H, (CH₃)₃CSi), 0.86 (s, 9 H,(CH₃)₃CSi), 0.11 (s, 6 H, (CH₃)₂Si), 0.04 (s, 6 H, (CH₃)₂Si).

N¹,N²-Bis-tert-butyloxycarbonyl-N²-acetyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidine(dG.03)

A solution of di-tert-butyldicarbonate (8.75 g, 40 mmol) in anhydrousCH₂Cl₂ (27 mL) was added to a solution of compound dG.02 (1.85 g, 3.44mmol), Et₃N (12.17 mL, 15.48 mmol), and DMAP (1.72 g, 14.10 mmol) inanhydrous CH₂Cl₂ (20 mL) under a N₂ atmosphere. The reaction mixture wasrefluxed for two hours, concentrated in vacuo, and purified by silicagel chromatography to yieldN¹,N²-bis-tert-butyloxy-carbonyl-N²-acetyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidinedG.03 (1.37 g, 54%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.25 (s, 1 H, H-8), 6.43 (t, 1 H, J=6.4 Hz,H-1′), 4.59 (m, 1 H, H-3′), 3.99 (m, 1 H, H-4′), 3.87 (m, 1 H, H-5′a),3.87 (m, 1 H, H-5′b), 2.60 (a, 3 H, CH₃CO), 2.52 (m, 1 H, H-2′a), 2.41(m, 1 H, H-2′b), 1.71 (s, 9 H, (CH₃)₃CO), 1.36 (s, 9H, (CH₃)₃CO), 0.92(s, 9 H, (CH₃)₃CSi), 0.91 (s, 9 H, (CH₃)₃CSi), 0.10 (s, 6 H, (CH₃)₂Si),0.09 (s, 6 H, (CH₃)₂Si).

N¹,N²-Bis-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidine(dG.04)

Compound dG.03 (1.31 g, 1.78 mmol) was treated with K₂CO₃ (1.23 g, 8.90mmol) in MeOH (20.5 mL) at room temperature for 30 minutes. The reactionwas concentrated in vacuo, dissolved in ethyl acetate (50 mL), andwashed twice with water (10 mL). The organic layer was dried with Na₂SO₄and concentrated in vacuo to giveN¹,N²-bis-tert-butyloxycarbonyl-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidinedG.04 (1.18 g, 95%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 9.82 (bs, 1 H, N—H), 8.24 (s, 1 H, H-8),6.27 (t, 1 H, J=6.6 Hz, H-1′), 4.71 (m, 1H, H-3′), 3.82 (m, 1 H, H-4′),3.79 (m, 1 H, H-5′a), 3.73 (m, 1 H, H-5′b), 2.95 (m, 1 H, H-2′a), 2.30(m, 1 H, H-2′b), 1.66 (s, 9 H, (CH₃)₃CO), 1.48 (s, 9 H, (CH₃)₃CO), 0.89(s, 9 H, (CH₃)₃CSi), 0.83 (s, 9 H, (CH₃)₃CSi), 0.11 (2 s, 6 H,(CH₃)₂Si), −0.01 (2 s, 6 H, (CH₃)₂Si).

N¹,N²-Bis-tert-butyloxycarbonyl-N²-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidine(dG.05)

NaH (22 mg, 0.93 mmol, dry) was added to a solution of compound dG.04(500 mg, 0.72 mmol) in anhydrous DMF (6 mL) at 0° C. and stirred for 30minutes under a N₂ atmosphere. A solution of 2-nitrobenzyl bromide (202mg, 0.94 mmol) in anhydrous DMF (3 mL) was added dropwise. The reactionwas stirred at 0° C. for one hour and then concentrated in vacuo. Ethylacetate (50 mL) was added, and the mixture was washed twice withsaturated NH₄Cl solution (20 mL). The combined aqueous layer wasextracted with ethyl acetate (20 mL), and the combined organic layer wasdried with Na₂SO₄, concentrated in vacuo, and purified by silica gelchromatography to yieldN¹,N²-bis-tert-butyloxycarbonyl-N²-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidinedG.05 (543 mg, 91%) as a white foam.

¹H NMR (400 MHz, CDCl₃): δ 8.12 (s, 1 H, H-8), 8.06 (m, 1 H, Ph-H), 7.68(m, 1 H, Ph-H), 7.56 (m, 1 H, Ph-H), 7.38 (m, 1 H, Ph-H), 6.41 (6, J=6.2Hz, 1 H, H-2′), 5.49 (s, 2 H, Ph-CH₂), 4.56 (m, 1 H, H-3′), 4.00 (m, 1H, H-4′), 3.80 (m, 2 H, H-5′), 2.52 (m, 1 H, H-2′a), 2.39 (m, 1 H,H-2′b), 1.54 (s, 9 H, (CH₃)₃CO), 1.45 (s, 9 H, (CH₃)₃CO), 0.92 (s, 18 H,(CH₃)₃CSi), 0.10 (s, 12 H, (CH₃)₂Si).

N²-(2-Nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidine(dG.06)

Silica gel 60 (4.5 g, 100-200 mesh, activated by heating to 50-60° C.under reduced pressure for 24 hours) was added to a solution of compounddG.05 (450 mg, 0.54 mmol) in CH₂Cl₂ (5 mL), and the mixture wasevaporated in vacuo to dryness. The residue was heated to 60-70° C.under reduced pressure for 48 hours, washed three times with MeOH (50mL), and filtered using a buchi funnel. The combined filtrate wasconcentrated in vacuo and purified by silica gel chromatography to yieldN2-(2-nitrobenzyl)-3′,5′-O-bis-tert-butyldimethylsilyl-2′-deoxyguanidinedG.06 (333 mg, 97%) as a white foam.

¹H NMR (400 MHz, DMSO-d₆): δ 10.85 (s, 1 H, N—H), 8.05 (m, 1 H, Ph-H),7.85 (s, 1 H, H-8), 7.69 (m, 1 H, Ph-H), 7.62 (m, 1 H, Ph-H), 7.54 (m, 1H, Ph-H), 7.10 (t, 1 H, N—H), 6.07 (t, 1 H, J=, H-1′), 4.78 (m, 2 H,Ph-CH₂), 4.40 (m, 1 H, H-3′), 3.79 (m, 1 H, H-4′), 3.62 (m, 2 H, H-5′),2.60 (m, 1 H, H-2′a), 2.15 (m, 1 H, H-2′b), 0.86 (s, 9H, (CH₃)₃CSi),0.85 (s, 9H, (CH₃)₃CSi), 0.06 (s, 6 H, (CH₃)₂Si), 0.01 (s, 6 H,(CH₃)₂Si).

N²-(2-Nitrobenzyl)-2′-deoxyguanidine (dG.07)

A solution of n-Bu₄NF (393 mg, 1.5 mmol) in THF (6 mL) was addeddropwise to a solution of compound dG.06 (313 mg, 0.5 mmol) in THF (12mL) at 0° C. The reaction mixture was stirred at 0° C. for one hour andthen at room temperature for two hours. The reaction was concentrated invacuo and purified by silica gel chromatography to yieldN²-(2-nitrobenzyl)-2′-deoxyguanidine dG.07 (165 mg, 83%) as a yellowfoam.

¹H NMR (400 MHz, DMSO-d₆): δ 10.82 (s, 1 H, D₂O exchangeable, N—H), 8.06(m, 1 H, Ph-H), 7.90 (s, 1 H, H-8), 7.72 (m, 1 H, Ph-H), 7.64 (m, 1 H,Ph-H), 7.55 (m, 1 H, Ph-H), 7.06 (t, 1 H, D₂O exchangeable, N—H), 6.08(t, 1 H, J=6.4 Hz, H-1′), 5.23 (d, 1 H, D₂O exchangeable, 3′-OH), 4.80(t, 3 H, among them 1H D₂O exchangeable, 5′-OH and Ph-CH₂), 4.25 (m, 1H, H-3′), 3.77 (m, 1 H, H-4′), 3.42 (m, 2 H, H-5′), 2.45 (m, 1 H,H-2′a), 2.12 (m, 1 H, H-2′b).

N²-(2-Nitrobenzyl)-2′-deoxyguanidine-5′-triphosphate (WW2p067)

POCl₃ (17 μL, 0.18 mmol) was added dropwise to a solution of compounddG.07 (50 mg, 0.12 mmol) in trimethylphosphate (0.5 mL) and maintainedat minus 20-30° C. for two hours. A solution of bis-tri-n-butylammoniumpyrophosphate (205 mg, 0.6 mmol) and tri-n-butylamine (120 μL) inanhydrous DMF (1.2 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred for one hour at room temperature and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to giveN²-(2-nitrobenzyl)-2′-deoxyguanidine-5′-triphosphate WW2p067 (52 mg,62%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.08 (d, 1 H, Ph-H), 8.04 (s, 1 H, H-8), 7.68(m, 2 H, Ph-H), 7.51 (t, 1 H, Ph-H), 6.27 (t, 1 H, J=6.8 Hz, H-1′), 4.63(m, 1 H, H-3′), 4.21 (m, 1 H, H-4′), 4.10 (m, 2 H, H-5′), 2.66 (m, 1 H,H-2′a), 2.41 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): 6-6.48 (d, J=15.7 Hz), −11.40 (d, J=15.2 Hz),−19.94 (br);

ToF-MS (ESI): For the molecular ion C₁₇H₁₉N₆O₁₅P₃Na [M-2H+Na]⁻, thecalculated mass was 663.0019, and the observed mass was 663.0143.

Synthesis of O⁶-(2-nitrobenzyl)-2′-deoxyguanosine-5′-triphosphate(WW2p077)

Scheme. Synthesis ofO⁶-(2-nitrobenzyl)-2′-deoxyguanosine-5′-triphosphate. (i)2-mesitylene-sulfonyl chloride, Et₃N, DMAP, anhydrous CH₂Cl₂, HMPA, roomtemperature, overnight, 98%; (ii) 2-nitrobenzyl alcohol, DABCO, DBU,molecular sieves, anhydrous 1,2-DME, 0° C., then gradually warmed toroom temperature, 24 hours, 77%; (iii) n-Bu₄NF, THF, room temperature,1.5 hours, 94%; (iv) POCl₃, (MeO)₃PO, minus 20-30° C., two hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, one hour,35%.

O⁶-(2-Mesitylenesulfonyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2-deoxyguanosine(dG.08)

2-Mesitylenesulfonyl chloride (510 mg, 2.34 mmol), Et₃N (0.56 mL, 4.0mmol), and DMAP (27 mg, 0.197 mmol) were added to a solution of compounddG.01 (500 mg, 1.0 mmol) in a mixture of hexmethylphosphoramide (1.5 mL)and anhydrous CH₂Cl₂ (7 mL). The reaction was stirred at roomtemperature overnight and then diluted with ethyl ether (25 mL). Theether solution was washed twice with a saturated solution of NaHCO₃ (10mL each) and then with brine (10 mL). The organic layer was dried overNa₂SO₄ and concentrated in vacuo to give a semi-solid, which wasdissolved in ethyl ether (2 mL) and gradually diluted with hexane (40mL). The precipitate was collected by filtration to yieldO⁶-(2-mesitylenesulfonyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosinedG.08 (737 mg, 98%).

¹H NMR (400 MHz, CDCl₃): δ 7.98 (s, 1 H, H-8), 6.98 (s, 2 H, Ph-H), 6.28(t, 1 H, J=6.5 Hz, H-1′), 4.84 (br s, 2 H, NH₂), 4.57 (m, 1 H, H-3′),3.97 (m, 1 H, H-4′), 3.78 (dd, 1 H, J=2.9 and 11.0 Hz, H-5′a), 3.75 (dd,1 H, J=2.9 and 11.0 Hz, H-5′b), 2.75 (s, 6 H, CH₃), 2.53 (m, 1 H,H-2′a), 2.34 (m, 1 H, H-2′b), 2.31 (s, 3 H, CH₃), 0.91 (s, 9 H,(CH₃)₃CSi), 0.90 (s, 9 H, (CH₃)₃CSi), 0.09 (s, 6 H, (CH₃)₂Si), 0.06 (s,6 H, (CH₃)₂Si).

O⁶-(2-Nitrobenzyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(dG.09)

DABCO (139 mg, 1.24 mmol) and 2-nitrobenzyl alcohol (474 mg, 3.10 mmol)were added to a solution of compound dG.08 (420 mg, 0.62 mmol) and 4{acute over (Å)} molecular sieves (200 mg) in anhydrous 1,2-DME (6.2 mL)at 0° C. The mixture was warmed to room temperature and stirred for 30minutes. DBU (139 μL, 0.93 mmol) was added, and the reaction was stirredat room temperature for 24 hours. Ethyl acetate (100 mL) was added, andthe organic layer was washed with water (20 mL) and brine (20 mL), driedover Na₂SO₄, concentrated in vacuo, and purified by silica gelchromatography to yieldO⁶-(2-nitrobenzyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosinedG.09 (300 mg, 77%).

¹H NMR (400 MHz, CDCl₃): δ 8.09 (dd, 1 H, J=1.1 and 8.2 Hz, Ph-H), 7.94(s, 1 H, H-8), 7.86 (d, 1 H, J=7.7 Hz, Ph-H), 7.60 (dt, 1 H, J=1.1 and7.7 Hz, Ph-H), 7.45 (dt, 1 H, J=1.0 and 8.2 Hz, Ph-H), 6.32 (t, 1 H,J=6.5 Hz, H-1′), 5.94 (s, 2H, NH₂), 4.89 (s, 2 H, PhCH₂), 4.59 (m, 1 H,H-3′), 3.98 (m, 1 H, H-4′), 3.81 (dd, 1 H, J=2.9 and 11.0 Hz, H-5′a),3.75 (dd, 1 H, J=2.9 and 11.0 Hz, H-5′b), 2.58 (m, 1 H, H-2′a), 2.37 (m,1 H, H-2′b), 0.91 (s, 18 H, (CH₃)₃CSi), 0.10 (s, 6 H, (CH₃)₂Si), 0.08(s, 6 H, (CH₃)₂Si).

O⁶-(2-Nitrobenzyl)-2′-deoxyguanosine (dG.10)

A solution of n-Bu₄NF (252 mg, 0.80 mmol) in THF (4 mL) was added to asolution of compound dG.09 (200 mg, 0.32 mmol) in THF (4 mL) at roomtemperature. The mixture was stirred for 1.5 hours, concentrated invacuo, and purified by silica gel chromatography to yieldO⁶-(2-nitrobenzyl)-2′-deoxyguanosine dG.10 (119 mg, 94%).

¹H NMR (400 MHz, DMSO-d₆): δ 8.16 (dd, 1 H, J=1.0 and 8.2, Hz, Ph-H),8.13 (s, 1 H, H-8), 7.79 (m, 2 H, Ph-H), 7.63 (m, 1 H, Ph-H), 6.49 (brs, 2 H, D₂O exchangeable, NH₂), 6.22 (dd, 1 H, J=6.1 and 7.7 Hz, H-1′),5.87 (s, 2 H, PhCH₂), 5.28 (br, 1 H, D₂O exchangeable, 5′-OH), 4.99 (br,1 H, D₂O exchangeable, 3′-OH), 4.35 (m, 1 H, H-3′), 3.82 (m, 1 H, H-4′),3.55 (m, 1 H, H-5′b), 3.52 (m, 1 H, H-5′a), 2.58 (m, 1 H, H-2′a), 2.23(m, 1 H, H-2′b).

O⁶-(2-Nitrobenzyl)-2′-deoxyguanosine-5′-triphosphate (WW2p077)

POCl₃ (14 μL, 0.1 mmol) was added dropwise to a solution of compounddG.10 (43 mg, 0.1 mmol) in trimethylphosphate (0.5 mL) and maintained atminus 20-30° C. for two hours. A solution of bis-tri-n-butylammoniumpyrophosphate (237 mg, 0.5 mmol) and tri-n-butylamine (100 μL) inanhydrous DMF (1.0 mL) was added. After five minutes of stirring,triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was added. Thereaction was stirred for one hour at room temperature and thenlyophilized to dryness. The residue was dissolved in water (10 mL),filtered, and purified by anion exchange chromatography using a QSepharose FF column (2.5×20 cm) with a linear gradient of NH₄HCO₃ (50 mMto 500 mM in 300 minutes) at a flow rate of 4.5 mL/min. The fractionscontaining triphosphate were combined and lyophilized to giveO⁶-(2-nitrobenzyl)-2′-deoxyguanosine-5′-triphosphate WW2p077 (24 mg,35%) as a white fluffy solid.

¹H NMR (400 MHz, D₂O): δ 8.21 (s, 1 H, H-8), 8.13 (d, J=8.2 Hz, 1 H,Ph-H), 7.83 (d, 1 H, J=7.8 Hz, Ph-H), 7.74 (t, 1 H, J=7.8 Hz, Ph-H),7.51 (t, J=7.8 Hz, 1 H, Ph-H), 6.35 (t, 1 H, J=6.8 Hz, H-1′), 5.86 (s, 2H, Ph-CH₂), 4.28 (m, 1 H, H-4′), 4.23 (m, 2 H, H-5′), 2.82 (m, 1 H,H-2′a), 2.57 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): δ −6.48 (br), −10.96 (br), −21.83 (br);

ToF-MS (ESI): For the molecular ion C₁₇H₁₉N₆O₁₅P₃Na [M-2H+Na]⁻, thecalculated mass was 663.0019, and the observed mass was 663.0228.

Synthesis of 6-ROX labeledO⁶-[4-(3-amino-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine-5′-triphosphate(WW2p121)

Scheme. Synthesis of 6-ROX labeledO⁶-[4-(3-amino-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine-5′-triphosphate.(i) 4-iodo-2-nitrobenzyl alcohol, DABCO, DBU, 4 {acute over (Å)}molecular sieves, anhydrous 1,2-DME, 0° C., then gradually warmed toroom temperature, 24 hours, 77%; (ii) n-Bu₄NF, THF, room temperature,1.5 hours, 62%; (iii) N-propargyltrifluoroacetamide, Pd(PPh₃)₄, CuI,Et₃N, anhydrous DMF, four hours, 42%; (iv) POCl₃, proton sponge,(MeO)₃PO, 0° C., one hour; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes;1 M HNEt₃HCO₃, one hour; NH₄OH, one hour; (v) 6-ROX-SE, 0.1 MNa₂CO₃/NaHCO₃ buffer (pH 9.2), one hour.

O⁶-(4-Iodo-2-nitrobenzyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2-deoxyguanosine(dG.11)

DABCO (90 mg, 0.80 mmol) and 4-iodo-2-nitrobenzyl alcohol (555 mg, 2.00mmol) were added to a solution of compound dG.08 (270 mg, 0.40 mmol) and4 {acute over (Å)} molecular sieves (129 mg) in anhydrous 1,2-DME (4 mL)at 0° C. The mixture was warmed to room temperature and stirred for 30minutes. DBU (91 μL, 0.60 mmol) was added, and the reaction was stirredat room temperature for 24 hours. Ethyl acetate (70 mL) was added, andthe organic solution was washed with water (10 mL) and brine (10 mL),dried over Na₂SO₄, concentrated in vacuo, and purified by silica gelchromatography to giveO⁶-(4-iodo-2-nitrobenzyl)-3′,5′-bis-O-(tert-butyldimethylsilyl)-2-deoxyguanosinedG.11 (233 mg, 77%).

¹H NMR (400 MHz, CDCl₃): δ 8.40 (d, 1 H, J=1.7 Hz, Ph-H), 7.94 (s, 1 H,H-8), 7.86 (dd, 1 H, J=1.7 and 8.3 Hz, Ph-H), 7.60 (d, 1 H, J=8.3 Hz,Ph-H), 6.31 (t, 1 H, J=6.5 Hz, H-1′), 5.86 (s, 2 H, NH₂), 4.87 (s, 2 H,PhCH₂), 4.59 (m, 1 H, H-3′), 3.98 (m, 2 H, H-4′), 3.82 (dd, AB, 1 H,J=4.2 and 11.2 Hz, H-5′a), 3.75 (dd, 1 H, J=4.2 and 11.2 Hz, H-5′b),2.57 (m, 1 H, H-2′a), 2.37 (m, 1 H, H-2′b), 0.91 (s, 18 H, (CH₃)₃CSi),0.10 (s, 6 H, (CH₃)₂Si), 0.08 (s, 6 H, (CH₃)₂Si).

O⁶-(4-Iodo-2-nitrobenzyl)-2-deoxyguanosine (dG.12)

A solution of n-Bu₄NF (291 mg, 0.924 mmol) in THF (2 mL) was added to asolution of compound dG.11 (233 mg, 0.31 mmol) in THF (4 mL) at roomtemperature. The mixture was stirred for 1.5 hours, concentrated invacuo, and purified by silica gel chromatography to giveO⁶-(4-iodo-2-nitrobenzyl)-2-deoxyguanosine dG.12 (101 mg, 62%).

¹H NMR (400 MHz, DMSO-d₆): δ 8.43 (d, 1 H, J=1.8 Hz, Ph-H), 8.16 (dd, 1H, J=1.8 and 8.2 Hz, Ph-H), 8.13 (s, 1 H, H-8), 7.52 (d, 1 H, J=8.2 Hz,Ph-H), 6.48 (br s, 2 H, D₂O exchangeable, NH₂), 6.22 (dd, 1 H, J=6.2 and7.7 Hz, H-1′), 5.79 (s, 2 H, PhCH₂), 5.27 (d, 1 H, D₂O exchangeable,5′-OH), 4.97 (t, 1 H, D₂O exchangeable, 3′-OH), 4.35 (m, 1 H, H-3′),3.82 (m, 1 H, H-4′), 3.52 (m, 2 H, H-5′a and H-5′b), 2.58 (m, 1 H,H-2′a), 2.22 (m, 1 H, H-2′b).

O⁶-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine(dG.13)

A solution of compound dG.12 (95 mg, 0.18 mmol),N-propargyltrifluoroacetylamide (82 mg, 0.53 mmol),tetrakis(triphenylphosphine)-palladium(0) (21 mg, 0.018 mmol), CuI (7mg, 0.036 mmol) and Et₃N (51 μL, 0.36 mmol) in anhydrous DMF (1.4 mL)was stirred at room temperature for four hours. CH₂Cl₂ (1 mL), methanol(1 mL), and NaHCO₃ (84 mg, 1 mmol) were added, and the mixture wasstirred for 20 minutes, concentrated in vacuo, and purified by silicagel column chromatography and preparative HPLC to giveO⁶-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosinedG.13 (42 mg, 42%).

¹H NMR (400 MHz, DMSO-d₆): δ 10.11 (br 1 H, NH), 8.16 (d, 1 H, J=1.7 Hz,Ph-H), 8.13 (s, 1 H, H-8), 7.86 (dd, 1 H, J=1.7 and 8.2 Hz, Ph-H), 7.75(d, 1 H, J=8.2 Hz, Ph-H), 6.50 (br s, 2 H, D₂O exchangeable, NH₂), 6.22(t, 1 H, J=6.4 Hz, H-1′), 5.87 (s, 2 H, PhCH₂), 5.27 (d, 1 H, D₂Oexchangeable, 5′-OH), 4.97 (t, 1 H, D₂O exchangeable, 3′-OH), 4.35 (m, 1H, H-3′), 4.33 (s, 2 H, CH₂), 3.82 (m, 2 H, H-4′), 3.55 (m, 1 H, H-5′b),3.51 (m, 1 H, H-5′a), 2.58 (m, 1 H, H-2′a), 2.23 (m, 1 H, H-2′b).

O⁶-[4-(3-Amino-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine-5′-triphosphate(dG.14)

Compound dG.13 (33 mg, 0.06 mmol) and proton sponge (26 mg, 0.12 mmol)were evaporated three times from anhydrous pyridine (3 mL) and dissolvedin trimethylphosphate (0.3 mL). POCl₃ (8 μL, 0.09 mmol) was added, andthe mixture was stirred for one hour at 0° C. A solution ofbis-tri-n-butylammonium pyrophosphate (142 mg, 0.3 mmol) andtri-n-butylamine (60 μL) in anhydrous DMF (0.6 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature, followed by the dropwise addition of concentrated ammoniumhydroxide (5 mL, 27%) at 0° C. The mixture was stirred for oneadditional hour at room temperature and then lyophilized to dryness. Theresidue was dissolved in water (10 mL), filtered, and purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yieldO⁶-[4-(3-amino-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine-5′-triphosphatedG.14. Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water(pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved using a linear gradient of 5-50% B for 20 minutes and then50-90% B for 10 minutes.

¹H NMR (400 MHz, D₂O): δ 8.28 (s, H-8), 8.26 (s, 1 H, Ph-H), 7.80 (d, 1H, J=8.0 Hz, Ph-H), 7.63 (d, 1 H, J=8.0 Hz, Ph-H), 6.38 (t, 1 H, J=6.4Hz, H-1′), 5.88 (br s, 2 H, Ph-CH₂), 4.29 (m, 3 H, H-4′, H-5′), 3.67 (s,2 H, CH₂), 2.80 (m, 1 H, H-2′a), 2.56 (m, 1 H, H-2′b);

³¹P NMR (162 MHz, D₂O): 6-5.85 (d, J=19.4 Hz), −10.98 (d, J=19.4 Hz),−21.78 (t, J=19.4 Hz);

ToF-MS (ESI): For the molecular ion C₂₀H₂₄N₇O₁₅P₃Na [M+Na]⁺, thecalculated mass was 718.0441, and the observed mass was 718.0600.

6-ROX LableledO⁶-[4-(3-Amino-1-propynyl)-2-nitrobenzyl]-2′-deoxyguanosine-5′-triphosphate(WW2p121)

A solution of 6-ROX-SE (0.3 mg, 0.47 μmol) in anhydrous DMSO (12 μL) wasadded to a solution of triphosphate dG.14 (0.36 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 0.6 mL) and incubated at room temperature for onehour. The reaction was purified with reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-ROX labeledtriphosphate WW2p121. Mobile phase: A, 100 mM TEAA in water (pH 7.0); B,100 mM TEAA in water/CH₃CN (30:70). HPLC purification was achieved usinga linear gradient of 5-50% B for 20 minutes and then 50-90% B for 10minutes. The concentration of WW2p121 was estimated by adsorptionspectroscopy using the extinction coefficient of the 6-ROX dye (i.e.,82,000 at 575 nm).

Synthesis ofO⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyguanosine-5′-triphosphate (WW2p143)

Scheme. Synthesis ofO⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyguanosine-5′-triphosphate. (i)1-(2-nitrophenyl)ethanol, PPh₃, DIAD, anhydrous THF, room temperature,six hours, 74%; (ii) n-Bu₄NF, THF, 0° C., then gradually warmed to roomtemperature, four hours, 64%; (iii) POCl₃, proton sponge, (MeO)₃PO, 0°C., two hours; (n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 MHNEt₃HCO₃, one hour; NH₄OH, 60° C., three hours.

O⁶-[1-(2-Nitrophenyl)ethyl]-N²-acetyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(dG.15)

A solution of compound dG.02 (108 mg, 0.2 mmol),1-(2-nitrophenyl)ethanol (33 mg, 0.23 mmol) and PPh₃ (79 mg, 0.3 mmol)in anhydrous THF (2 mL) was treated with diisopropyl azodicarboxylate(DIAD, 59 μL, 0.3 mmol) and stirred for six hours at room temperature.The mixture was diluted with CH₂Cl₂ (20 mL), washed once with saturatedNH₄Cl solution (10 mL), dried over Na₂SO₄, concentrated, and purified bysilica gel chromatography to yieldO⁶-[1-(2-nitrophenyl)ethyl]-N²-acetyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosinedG.15 (102 mg, 74%, 1:1 mixture of diastereomers) as a yellow foam.

¹H NMR (400 MHz, CDCl₃) for diastereomers: 68.13 and 8.12 (2 s, 1 H,H-8). 7.89 (d, 1 H, J=8.0 Hz, Ph-H), 7.83 (m, 1 H, Ph-H), 7.74 (br s., 1H, NH), 7.57 (t, 1 H, J=7.2 Hz, Ph-H), 7.40 (t, 1 H, J=8.0 Hz, Ph-H),6.69 (m, 1 H, PhCH), 6.36 (t, 1 H, J=6.3 Hz, H-1′), 4.56 (m, 1 H, H-3′),3.98 (m, 1 H, H-4′), 3.82 (m, 1 H, H-5′a), 3.76 (m, 1 H, H-5′b), 2.50(m, 1 H, H-2′a), 2.41 (m, 4 H, H-2′b and CH₃CO), 1.88 (2 d, J=6.5 Hz,CH₃), 0.91 (s, 18 H, (CH₃)₃CSi), 0.10 (s, 6 H, (CH₃)₂Si), 0.09 (s, 6 H,(CH₃)₂Si).

O⁶-[1-(2-Nitrophenyl)ethyl]-N²-acetyl-2′-deoxyguanosine (dG.16)

A solution of n-Bu₄NF (95 mg, 0.36 mmol) in THF (2 mL) was added to asolution of compound dG.15 (100 mg, 0.15 mmol) in THF (5 mL) at 0° C.The mixture was gradually warmed to room temperature and stirred forfour hours. Silica gel (500 mg, 60-200 mesh) was added, and the mixturewas evaporated in vacuo. The residue was purified by silica gelchromatography to yieldO⁶-[1-(2-nitrophenyl)ethyl]-N²-acetyl-2′-deoxyguanosine dG.16 (43 mg,64%, 1:1 mixture of diastereomers).

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.19 and 10.18 (2 s, 1H, D₂O exchangeable, NH), 8.43 (2 s, 1 H, H-8), 8.04 (d, J=8.2 Hz,Ph-H), 7.79-7.74 (m, 2 H, Ph-H), 7.56 (t, 1 H, J=8.1 Hz, Ph-H), 6.84 (m,1 H, PhCH), 6.26 (t, 1 H, J=6.8 Hz, H-1′), 5.29 (2 d, 1 H, D₂Oexchangeable, 5′-OH), 4.88 (m, 1 H, D₂O exchangeable, 3′-OH), 4.39 (m, 1H, H-3′), 3.82 (m, 1 H, H-4′), 3.56 (m, 1 H, H-5′b), 3.51 (m, 1 H,H-5′a), 2.56 (m, 1 H, H-2′a), 2.23 (m, 1 H, H-2′b), 2.12 (s, 3 H,CH₃CO), 1.79 (2 d, J=6.4 Hz, CH₃);

ToF-MS (ESI): For the molecular ion C₂₀H₂₁N₆O₇ [M-H]⁻, the calculatedmass was 457.1472, and the observed mass was 457.1392.

O⁶-[1-(2-Nitrophenyl)ethyl]-2′-deoxyguanosine-5′-triphosphate (WW2p143)

Compound dG.16 (25 mg, 0.055 mmol) and proton sponge (23 mg, 0.11 mmol)were evaporated three times from anhydrous pyridine (2 mL) and dissolvedin trimethylphosphate (0.3 mL). POCl₃ (8 μL, 0.08 mmol) was added, andthe mixture was stirred for two hours at 0° C. A solution ofbis-tri-n-butylammonium pyrophosphate (130 mg, 0.28 mmol) andtri-n-butylamine (55 μL) in anhydrous DMF (0.55 mL) was added. Afterfive minutes of stirring, triethylammonium bicarbonate buffer (1 M, pH7.5; 10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and part of the solution was purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yieldO⁶-[1-(2-nitrophenyl)ethyl]-N²-acetyl-2′-deoxyguanosine-5′-triphosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved using a linear gradient of 5-50% B for 40 minutes and then50-90% B for 10 minutes. The purified triphosphate was then treated withconcentrated ammonium hydroxide (2 mL, 27%) at 60° C. for three hours.Purification using reverse-phase HPLC was performed as described aboveto yield O⁶-[1-(2-nitrophenyl)ethyl]-2′-deoxyguanosine-5′-triphosphateWW2p143 (1:1 mixture of diastereomers).

¹H NMR (400 MHz, D₂O) for diastereomers: 8.26 and 8.25 (2 s, 1 H, H-8),8.02 (d, J=8.2 Hz, Ph-H), 7.88 (d, 1 H, J=7.8 Hz, Ph-H), 7.72 (t, 1 H,J=7.6 Hz, Ph-H), 7.53 (t, 1 H, J=8.2 Hz, Ph-H), 6.71 (m, 1 H, PhCH),6.34 (t, 1 H, J=6.8 Hz, H-1′), 4.25-4.16 (m, 3 H, H-4′ and H-5′), 2.80(m, 1 H, H-2′a), 2.51 (m, 1 H, H-2′b), 1.86 (d, 1 H, J=6.4 Hz, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: 6-5.18 (d, J=20.4 Hz), −10.25(d, J=19.3 Hz), −21.07 (t, J=19.8 Hz);

ToF-MS (ESI): For the molecular ion C₁₈H₂₂N₆O₁₅P₃ [M-H]⁻, the calculatedmass was 655.0356, and the observed mass was 655.0430.

Synthesis of 6-ROX LabeledO⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p008)

Scheme. Synthesis of 6-ROX labeledO⁶-{1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate.(i) 1-(4-iodo-2-nitrophenyl)ethanol, PPh₃, DIAD, anhydrous THF, roomtemperature, overnight, 76%; (ii) n-Bu₄NF, THF, 0° C., then graduallywarmed to room temperature, TWO hours, 52%; (iii)N-propargyltrifluoroacetamide, Pd(PPh₃)₄, CuI, Et₃N, anhydrous DMF, fourhours, 96%; (iv) POCl₃, proton sponge, (MeO)₃PO, 0° C., two hours;(n-Bu₃NH)₂H₂P₂O₇, n-Bu₃N, DMF, five minutes; 1 M HNEt₃HCO₃, one hour;NH₄OH, 60° C., six hours; (v) 6-ROX-SE, 0.1 M Na₂CO₃/NaHCO₃ buffer (pH9.2), one hour.

O⁶-[1-(4-Iodo-2-nitrophenyl)ethyl]-N²-acetyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosine(dG.17)

A solution of compound dG.02 (146 mg, 0.27 mmol),1-(4-iodo-2-nitrophenyl)ethanol (79 mg, 0.27 mmol) and PPh₃ (106 mg, 0.4mmol) in anhydrous THF (2 mL) was treated with diisopropylazodicarboxylate (DIAD, 79 μL, 0.4 mmol) and stirred overnight at roomtemperature. The mixture was diluted with CH₂Cl₂ (20 mL), washed oncewith saturated NH₄Cl solution (10 mL), dried over Na₂SO₄, concentrated,and purified by silica gel chromatography to yieldO⁶-[1-(4-iodo-2-nitrophenyl)ethyl]-N²-acetyl-3′,5′-bis-O-(tert-butyldimethylsilyl)-2′-deoxyguanosinedG.17 (168 mg, 76%, 1:1 mixture of diastereomers) as a yellow foam.

¹H NMR (400 MHz, CDCl₃) for diastereomers: δ 8.20 (s, 1 H, Ph-H), 8.13and 8.12 (2 s, 1 H, H-8), 7.87 (d, 1 H, J=8.0 Hz, Ph-H), 7.73 (br s, 1H, NH), 7.55 (d, 1 H, J=8.0 Hz, Ph-H), 6.62 (m, 1 H, PhCH), 6.35 (t, 1H, J=6.5 Hz, H-1′), 4.56 (m, 1 H, H-3′), 3.98 (m, 1 H, H-4′), 3.82 (m, 1H, H-5′a), 3.77 (m, 1 H, H-5′b), 2.50 (m, 1 H, H-2′a), 2.41 (m, 4 H,H-2′b and CH₃CO), 1.85 (d, J=6.4 Hz, CH₃), 0.91 (s, 18 H, (CH₃)₃CSi),0.09 (2 s, 12 H, (CH₃)₂Si).

O⁶-[1-(4-Iodo-2-nitrophenyl)ethyl]-N²-acetyl-2′-deoxyguanosine (dG.18)

A solution of n-Bu₄NF (125 mg, 0.48 mmol) in THF (1.5 mL) was added to asolution of compound dG.17 (155 mg, 0.19 mmol) in THF (2 mL) at 0° C.The reaction mixture was gradually warmed to room temperature andstirred for two hours. Silica gel 60 (1 g, 60-200 mesh) was added, andthe mixture was evaporated in vacuo to dryness. The residue was purifiedby silica gel column chromatography to yieldO⁶-[1-(4-iodo-2-nitrophenyl)ethyl]-N²-acetyl-2′-deoxyguanosine dG.18 (58mg, 52%, 1:1 mixture of diastereomers) as a white foam.

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.21 and 10.20 (2 s, 1H, D₂O exchangeable, NH), 8.43 (2 s, 1 H, H-8), 8.34 (2 s, Ph-H), 8.08(2 d, 1 H, J=8.3 Hz, Ph-H), 7.54 (2 d, 1 H, J=8.3 Hz, Ph-H), 6.75 (m, 1H, PhCH), 6.27 (t, 1 H, J=6.4 Hz, H-1′), 5.30 (m, 1 H, D₂O exchangeable,5′-OH), 4.89 (m, 1 H, D₂O exchangeable, 3′-OH), 4.39 (m, 1 H, H-3′),3.82 (m, 1 H, H-4′), 3.56 (m, 1 H, H-5′b), 3.50 (m, 1 H, H-5′a), 2.60(m, 1 H, H-2′a), 2.22 (m, 1 H, H-2′b), 2.12 (s, 3 H, CH₃CO), 1.75 (2 d,J=6.4 Hz, CH₃).

O⁶-{1-[4-(3-Trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-N²-acetyl-2′-deoxyguanosine(dG.19)

A solution of compound dG.18 (58 mg, 0.1 mmol), N-propargyltrifluoroacetylamide (45 mg, 0.3 mmol),tetrakis(triphenylphosphine)-palladium(0) (11.5 mg, 0.01 mmol),copper(I) iodide (3.8 mg, 0.02 mmol) and triethylamine (27 μL, 0.19mmol) was stirred at room temperature for four hours. Methanol (1 mL),CH₂Cl₂ (1 mL), and sodium bicarbonate (80 mg, 0.95 mmol) were added, andthe mixture was stirred for an additional half hour, then concentratedin vacuo and purified by column chromatography on silica gel to yieldO⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-N²-acetyl-2′-deoxyguanosinedG.19 (58 mg, 96%, 1:1 mixture of diastereomers).

¹H NMR (400 MHz, DMSO-d₆) for diastereomers: δ 10.21 and 10.20 (2 s. 1H, D₂O exchangeable, NH), 10.08 (br, 1 H, NHCOCF₃), 8.43 (2 s, 1 H,H-8), 8.06 (s, 1 H, Ph-H), 7.77 (m, 2 H, Ph-H), 6.78 (m, 1 H, PhCH),6.28 (t, 1 H, J=6.4 Hz, H-1′), 5.29 (2 d, 1 H, D₂O exchangeable, 5′-OH),4.89 (t, 1 H, D₂O exchangeable, 3′-OH), 4.39 (m, 1 H, H-3′), 4.29 (d, 2H, CH₂), 3.82 (m, 2 H, H-4′), 3.50 (m, 1 H, H-5′a), 3.44 (m, 1 H,H-5′b), 2.67 (m, 1 H, H-2′a), 2.23 (m, 1 H, H-2′b), 2.11 (s, 3 H, CH₃),1.78 (d, 3 H, J=6.4 Hz, CH₃);

O⁶-{1-[4-(3-Amido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(dG.20)

Compound dG.19 (44 mg, 0.07 mmol) and proton sponge (30 mg, 0.14 mmol)were evaporated three times from anhydrous pyridine (3 mL) and dissolvedin trimethylphosphate (0.5 mL). POCl₃ (10 μL, 0.11 mmol) was added, andthe mixture was stirred for three hours at 0° C. A solution ofbis-tri-n-butylammonium pyrophosphate (166 mg, 0.35 mmol) andtri-n-butylamine (70 μL) in anhydrous DMF (0.7 mL) was added. After fiveminutes of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;10 mL) was added. The reaction was stirred for one hour at roomtemperature and then lyophilized to dryness. The residue was dissolvedin water (10 mL), filtered, and part of the solution was purified withreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yieldO⁶-{1-[4-(3-trifluoroacetamido-1-propynyl)-2-nitrophenyl]ethyl}-N²-acetyl-2′-deoxyguanosine-5′-triphosphate.Mobile phase: A, 100 mM triethylammonium acetate (TEAA) in water (pH7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLC purification wasachieved using a linear gradient of 5-50% B for 20 minutes and then50-90% B for 10 minutes. The purified triphosphate was then treated withconcentrated ammonium hydroxide (2 mL, 27%) at 60° C. for six hours toyieldO⁶-{1-[4-(3-amido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphatedG.20 (1:1 mixture of diastereomers).

¹H NMR (400 MHz, D₂O) for diastereomers: 8.21 and 8.20 (2 s, 1 H, H-8),7.85 and 7.75 (2 s, 1 H, Ph-H), 7.64 (m, 1 H, Ph-H), 7.45 (m, 1 H,Ph-H), 6.53 (m, 1 H, PhCH), 6.28 (m, 1 H, H-1′), 4.23-4.12 (m, 3 H, H-4′and H-5′), 3.97 (s, 2 H, CH₂), 2.70 (m, 1 H, H-2′a), 2.50 (m, 1 H,H-2′b), 1.74 (m, 1 H, CH₃);

³¹P NMR (162 MHz, D₂O) for diastereomers: 6-5.53 (d, J=20.1 Hz), −10.50(d, J=19.3 Hz), −21.29 (t, J=19.8 Hz);

ToF-MS (ESI): For the molecular ion C₂₁H₂₅N₇O₁₅P₃ [M-H]⁻, the calculatedmass was 708.0622, and the observed mass was 708.0609.

6-ROX LableledO⁶-{1-[4-(3-Amido-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p008)

A solution of 6-ROX-SE (3 mg, 4.7 μmol) in anhydrous DMSO (120 μL) wasadded to a solution of triphosphate dG.20 (1.45 μmol) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 0.3 mL) and incubated at room temperature for onehour. The reaction was purified with reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-ROX labeledtriphosphate WW3p008. Mobile phase: A, 100 mM TEAA in water (pH 7.0); B,100 mM TEAA in water/CH₃CN (30:70). HPLC purification was achieved usinga linear gradient of 5-50% B for 20 minutes and then 50-90% B for 10minutes. The concentration of WW3p008 was estimated by adsorptionspectroscopy using the extinction coefficient of the 6-ROX dye (i.e.,82,000 at 575 nm).

Separation of the Two Diastereoisomers ofO⁶-{1-[4-(3-amino-1-propynyl)-2-nitro-phenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(dG.20 ds1 and dG.20 ds2)

Separation of the two diastereoisomers of dG.20 was performed byreverse-phase HPLC using a Perkin Elmer OD-300 C₁₈ column (4.6×250 mm)to yield O⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosinetriphosphate dG.20 ds1 (single diastereoisomer, absolute configurationnot determined) and O⁶-{(S orR)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosinetriphosphate dG.20 ds2 (single diastereoisomer, absolute configurationnot determined). Mobile phase: A, 100 mM triethylammonium acetate (TEAA)in water (pH 7.0); B, 100 mM TEAA in water/CH₃CN (30:70). HPLCpurification was achieved using a linear gradient of 5-25% B for 70minutes and then 25-50% B for 30 minutes.

Synthesis of 6-ROX labeled Single Diastereoisomer O⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p037)

Scheme. Synthesis of 6-ROX labeled single diastereoisomer O⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate.(i) 6-ROX-SE, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour.

6-ROX Labeled Single Diastereoisomer O⁶-{(R orS)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p037)

A solution of 6-ROX-SE (1.5 mg, 2.38 μmol) in anhydrous DMSO (120 μL)was added to a solution of triphosphate dG.20 ds1 (0.67 μmol, singlediastereoisomer, absolute configuration not determined) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 150 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-ROX labeled singlediastereoisomer triphosphate WW3p037. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved using a lineargradient of 5-50% B for 20 minutes and then 50-90% B for 20 minutes. Theconcentration of WW3p037 was estimated by absorption spectroscopy usingthe extinction coefficient of the 6-ROX dye (i.e., 82,000 at 575 nm).

Synthesis of 6-ROX Labeled Single Diastereoisomer O⁶-{(S orR)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p039)

Scheme. Synthesis of 6-ROX labeled single diastereoisomer O⁶-{(S orR)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate.(i) 6-ROX-SE, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour.

6-ROX Labeled Single Diastereoisomer O⁶-{(S orR)-1-[4-(3-amino-1-propynyl)-2-nitrophenyl]ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p039)

A solution of 6-ROX-SE (2.5 mg, 3.96 μmol) in anhydrous DMSO (200 μL)was added to a solution of triphosphate dG.20 ds2 (0.97 μmol, singlediastereoisomer, absolute configuration not determined) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 150 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-ROX labeled singlediastereoisomer triphosphate WW3p039. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved using a lineargradient of 5-50% B for 20 minutes and then 50-90% B for 20 minutes. Theconcentration of WW3p039 was estimated by adsorption spectroscopy usingthe extinction coefficient of the 6-ROX dye (i.e., 82,000 at 575 nm).

Synthesis of 6-ROX Labeled Single Diastereoisomer O⁶-{(R orS)-1-{4-[3-(6-amino-caproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p041)

Scheme. Synthesis of 6-ROX labeled single diastereoisomer O⁶-{(R orS)-1-{4-[3-(6-aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyguanosinetriphosphate. (i) 6-N-(trifluoroacetyl)aminocaproic acid N-succinimidylester, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour; NH₄OH, one hour; (ii)6-ROX-SE, 0.1 M NaHCO₃/Na₂CO₃, pH 9.2, one hour.

O⁶-{(R orS)-1-{4-[3-(6-Aminocaproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyguanosine-5′-triphosphate(Single Diastereoisomer dG.21 ds1)

A solution of 6-N-(trifluoroacetyl)aminocaproic acid N-succinimidylester (1.0 mg, 3.08 μmol) in anhydrous DMSO (20 μL) was added to asolution of triphosphate dG.20 ds1 (0.89 μmol, single diastereoisomer,absolute configuration not determined) in Na₂CO₃/NaHCO₃ buffer (0.1 M,pH 9.2; 200 μL) and incubated at room temperature for one hour.Concentrated ammonium hydroxide (25% aq., 0.5 mL) was added, and themixture was incubated at room temperature for another hour. The reactionwas purified by reverse-phase HPLC using a Perkin Elmer OD-300 C₁₈column (4.6×250 mm) to yield the triphosphate dG.21 ds1 (singlediastereoisomer, absolute configuration not determined). Mobile phase:A, 100 mM triethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mMTEAA in water/CH₃CN (30:70). HPLC purification was achieved using alinear gradient of 5-50% B for 20 minutes and then 50-90% B for 10minutes.

Synthesis of 6-ROX Labeled Single Diastereoisomer O⁶-{(R orS)-1-{4-[3-(6-amino-caproyl)amino-1-propynyl]-2-nitrophenyl}ethyl}-2′-deoxyguanosine-5′-triphosphate(WW3p041)

A solution of 6-ROX-SE (1.5 mg, 2.34 μmol) in anhydrous DMSO (120 μL)was added to a solution of triphosphate dG.21 ds1 (0.59 μmol, singlediastereoisomer, absolute configuration not determined) in Na₂CO₃/NaHCO₃buffer (0.1 M, pH 9.2; 200 μL) and incubated at room temperature for onehour. The reaction was purified by reverse-phase HPLC using a PerkinElmer OD-300 C₁₈ column (4.6×250 mm) to yield the 6-ROX labeled singlediastereoisomer triphosphate WW3p041. Mobile phase: A, 100 mMtriethylammonium acetate (TEAA) in water (pH 7.0); B, 100 mM TEAA inwater/CH₃CN (30:70). HPLC purification was achieved using a lineargradient of 5-50% B for 20 minutes and then 50-90% B for 20 minutes. Theconcentration of WW3p041 was estimated by adsorption spectroscopy usingthe extinction coefficient of the 6-ROX dye (i.e., 82,000 at 575 nm).

Example 5 Polymerase End-Point (PEP) Assays

Numerous groups have employed qualitative, Sanger-based assays toestimate relative incorporation efficiencies of terminating nucleotideanalogs compared to their natural nucleotide counterparts. Althoughuseful, the ability to assay modified nucleotide analogs in the absenceof natural nucleotides is not feasible using these assays. These twolimitations led to a quantitative, polymerase end-point (PEP) assay,which is utilized in a high-throughput manner for screening numerousmodified nucleotides against a number of well-characterized,commercially available polymerases. Identification of lead compoundswith a specific DNA polymerase could then be prioritized for furtherkinetic studies. The PEP assay is designed with the polymeraseconcentration in excess of the primer/template complex (i.e., thiscomplex is fully bounded with polymerase at the start of the titration),thereby limiting reaction to nucleotide binding and nucleotyl couplingsteps. Limiting amounts of the desired nucleotide are then titratedacross the appropriate concentration range (generally three orders ofmagnitude) to observe extension of a dye-labeled primer by gelelectrophoresis. The end-point concentration is determined from asemi-log plot where the number of moles of substrate and product areequal, called the IC₅₀ (i.e., incorporation concentration at 50%) value.

For all polymerases evaluated in this study, 40 nM of oligo-template(5′-TACGGAGCA-GTACTGGCCGTCGTTTTACA (SEQ ID NO: 1), interrogation base isunderlined and bolded) was annealed to 5 nM BODIPY-FL labeled primer(5′-TTGTAAAACGACGGCCAGT (SEQ ID NO: 2)) in 1×ThermoPol buffer (20 mMTris-HCl, pH 8.8; 10 mM (NH₄)₂SO₄; 10 mM KCl; 2 mM MgSO₄; 0.1% TritonX-100, New England BioLabs) at 80° C. for 30 seconds, 57° C. for 30seconds, and then cooled to 4° C. The primer/template complex is thendiluted by one-half (i.e., its final concentration is 2.5 nM in a volumeof 10 μL) by the addition of DNA polymerase, nucleotide analog, andThermoPol buffer. This defines the lower limit of the IC₅₀ value fornucleotide titrations to 1.25 nM (i.e., [primer]=[primer+1]). Polymerasereactions were incubated at their appropriate temperature for 10minutes, then cooled to 4° C. and quenched with 10 μL of stop solution(98% deionized formamide; 10 mM Na₂EDTA, pH 8.0; 25 mg/mL Blue Dextran,MW 2,000,000). Stopped reactions were heated to 90° C. for 30 seconds,and then placed on ice. The extension products were analyzed on a 10%Long Ranger (Cambrex) polyacrylamide gel using an AB model 377 DNAsequencer, and the quantitative data are displayed as a linear-log plotof product formation versus compound concentration. The PEP assays wereperformed in triplicate for each DNA polymerase/nucleotide analogcombination to calculate its IC₅₀ values±one standard deviation.

The number of activity units for eight commercially available,3′-exonuclease deficient (3′-exo-) DNA polymerases was first determinedby titration with 2′-deoxyadenosine triphosphate (dATP, concentrationrange from 0.1 nM to 100 nM) with the goal of reaching the PEP IC₅₀limit of 1.25 nM (data not shown). In general, increasing the number ofunits reduced the IC₅₀ values for dATP towards this limit, withexception with Taq, THERMINATOR™, and THERMINATOR™ II. For theseenzymes, there was an increase in IC₅₀ values for dATP with increasingenzyme concentration, which was not investigated further and presumed tobe due to a direct relationship with increasing enzyme concentration andphosphorolysis. For these cases, the number of units used for subsequentPEP assays were those activity units that gave the lowest IC₅₀ valuesfor dATP.

Modified Nucleotide Titrations:

WW1p129 and ddATP were then titrated using the PEP assay with the eightDNA polymerases (unit activities previously defined) in theconcentration range of either 0.1 nM to 100 nM, 1 nM to 1 μM, 10 nM to10 μM, or 100 nM to 100 μM (see Table 1). UV-light sensitive compoundswere handled at all times in low light conditions to minimize conversionto dATP. These data show in all cases, except that for AMPLITAQ® DNApolymerase, FS, that WW1p129 is incorporated more efficiently (i.e.,lower IC₅₀ value) than ddATP.

TABLE 1 Summary of PEP assay result for WW1p129 using eight differentDNA polymerases IC₅₀ values DNA polymerase dATP WW1p129 ddATP Bst: 65°C. 1.2 ± 0.1 nM 21 ± 3 nM 0.37 ± 0.03 μM Klenow(3′-exo-): 1.6 ± 0.1 nM4.3 ± 0.2 nM 29 ± 5 nM 37° C. Taq: 68° C. 5.5 ± 0.5 nM 2.1 ± 0.2 μm 12.6± 0.9 μm Taq FS: 68° C. 5.3 ± 0.1 nM 0.89 ± 0.06 μm 3.3 ± 0.1 nmTherminator: 2.3 ± 0.3 nM 3.1 ± 0.4 nM 9.7 ± 1.1 nM 75° C. TherminatorII: 4.4 ± 0.6 nM 7.8 ± 0.7 nM 0.23 ± 0.03 μM 75° C. Vent(3′-exo-): 1.6 ±0.2 nM 2.1 ± 0.2 nM 0.55 ± 0.04 μM 72° C. DeepVent(3′-exo-): 2.8 ± 0.2nM 11.0 ± 0.6 nM 3.4 ± 0.4 μM 72° C.

PEP assays have also been performed using a number of photocleavableterminating nucleotides using Bst polymerase and Therminator DNApolymerase (Table 2), which shows the compounds are effectivelyincorporated. The data in the Table 2 suggests that the compoundsaccording to the invention are excellent substrates.

TABLE 2 Comparison of IC50 values with Bst and Therminator polymerasescompound Bst Therminator WW1p129 39 ± 9 nm 2.5 ± 0.4 nm VL3p03085 138 ±35 nm 1.1 ± 0.1 nm WW2p044 57 ± 11 nm 1.8 ± 0.2 nm WW2p077 3.8 ± 0.2micromolar 6.9 ± 0.5 nm WW2p050 n/a 4.4 ± 0.6 nm WW2p075 n/a 3.8 ± 1.1nm WW2p080 n/a 3.0 ± 0.6 nm WW2p121 n/a 6.3 ± 0.4 nm

All patents and patent publications referred to herein are herebyincorporated by reference. Certain modifications and improvements willoccur to those skilled in the art upon a reading of the foregoingdescription. It should be understood that all such modifications andimprovements have been deleted herein for the sake of conciseness andreadability but are properly within the scope of the following claims.

1. A compound selected from the group consisting of:

wherein R₁═H, monophosphate, diphosphate or triphosphate, R₂═H or OH, R₃and R₄ are each independently selected from the group consisting of H, aC₁-C₁₂ straight chain or branched alkyl, a C₂-C₁₂ straight chain orbranched alkenyl or polyenyl, a C₂-C₁₂ straight chain or branchedalkynyl or polyalkynyl, and an aromatic group, with the proviso that atleast one of R₃ and R₄ is H, R₅, R₆, R₇, and R₈ are each independentlyselected from the group consisting of H, OCH₃, NO₂, CN, a halide, aC₁-C₁₂ straight chain or branched alkyl, a C₂-C₁₂ straight chain orbranched alkenyl or polyenyl, a C₂-C₁₂ straight chain or branchedalkynyl or polyalkynyl, and an aromatic group, with the proviso that oneof R₅, R₆, R₇, and R₈ is a linker group of the structure —C≡CCH₂NH₂,—C≡CCH₂NHC(O)(CH₂)₅NH₂,

wherein X═CH₂, CH═CH, C≡C, O, S, or NH, Y=CH₂, O, or NH, n=an integerfrom 0-12; m=an integer from 0-12, and Dye=a fluorophore.
 2. A compoundaccording to claim 1, wherein R₃ and R₄ are each independently selectedfrom the group consisting of H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, isopropyl,tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl, with theproviso that at least one of R₃ and R₄ is H.
 3. A compound according toclaim 1, wherein R₃ or R₄ are each independently selected from the groupconsisting of H, alkyl and aromatic groups optionally containing atleast one heteroatom in the alkyl or aromatic groups, and furtherwherein the aromatic group may optionally be an aryl or polycyclicgroup, with the proviso that at least one of R₃ and R₄ is H.
 4. Thecompound of claim 2, wherein R₃ or R₄ is —CH₃ or isopropyl.
 5. Thecompound of claim 1, further defined as:


6. The compound of claim 5, further defined as:


7. The compound of claim 1, further defined as:

or a stereoisomer thereof.
 8. The compound of claim 1, further definedas:

or a stereoisomer thereof.
 9. The compound of claim 1, further definedas:


10. The compound of claim 1, further defined as:

or a stereoisomer thereof.
 11. The compound of claim 1, further definedas:

or a stereoisomer thereof.
 12. The compound of claim 1, further definedas:


13. The compound of claim 1, further defined as:


14. The compound of claim 1, further defined as:

or a stereoisomer thereof.
 15. The compound of claim 1, further definedas:

or a stereoisomer thereof.
 16. The compound of claim 1, further definedas:

or a stereoisomer thereof.
 17. The compound of claim 1, further definedas:


18. The compound of claim 1, further defined as:


19. The compound of claim 1, further defined as a compound of formula(I).
 20. The compound of claim 1, further defined as a compound offormula (II).
 21. The compound of claim 1, further defined as a compoundof formula (III).
 22. The compound of claim 1, further defined as acompound of formula (IV).
 23. The compound of claim 1, further definedas a compound of formula (V).
 24. The compound of claim 1, furtherdefined as a compound of formula (VI).
 25. The compound of claim 1,further defined as a compound of formula (VII).
 26. A compound accordingto any one of claims 19-25, wherein R₃ and R₄ are each independentlyselected from the group consisting of H, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃,isopropyl, tert-butyl, phenyl, 2-nitrophenyl, and 2,6-dinitrophenyl,with the proviso that at least one of R₃ and R₄ is H.
 27. A compoundaccording to any one of claims 19-25, wherein R₃ or R₄ are eachindependently selected from the group consisting of H, alkyl andaromatic groups optionally containing at least one heteroatom in thealkyl or aromatic groups, and further wherein the aromatic group mayoptionally be an aryl or polycyclic group, with the proviso that atleast one of R₃ and R₄ is H.
 28. A compound according to any one ofclaims 19-25, wherein R₃ or R₄ is —CH₃ or isopropyl.